Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The quenching of fluorescence (up to 98%) by anti-fluorescein antibodies is well documented in the literature. Here we report a system where, instead of quenching, bifluorophoric molecules are designed to increase in fluorescence upon binding by an anti-fluorescein antibody. Bifluorophoric molecules are made of fluorescein (F) linked to tetramethylrhodamine (T) via varying numbers of methylene units, denoted as F-(CH(2))(n)-T. These F-(CH(2))(n)-T conjugates are almost nonfluorescent when free in solution due to intramolecular dimerization and stacking. Upon binding to an anti-fluorescein antibody, however, up to 110-fold increase in fluorescence was observed from the rhodamine moiety. This increase is believed to result from intramolecular dimer dissociation that dequenches the rhodamine fluorescence. Fluorescein fluorescence, on the other hand, remains quenched due to binding and intramolecular resonance energy transfer. Moreover, the excitation wavelength was at the absorption maxima of fluorescein, giving a Stoke's shift of about 90 nm. This system couples directly molecular recognition with a concurrent increase in fluorescence emission, obviating wash and incubation steps required by most assays. It is an important molecular reporter system for developing homogeneous assays.
J Mol Recognit
PMID:Bifluorophoric molecules as fluorescent beacons for antibody-antigen binding. 1244 9

EnvZ is a sensory histidine kinase in Escherichia coli to regulate the phosphorylation of OmpR, its cognate response regulator, required for the expression of genes for outer membrane porin proteins. Here, we re-examined the recent paper Mattison and Kenney, in which the authors reported that phosphorylated OmpR (OmpR-P) is unable to bind to EnvZ, thus casting doubts on the role of the EnvZ phosphatase activity in vivo. Using an identical method, the Kd value for the interaction of the fluorescein-labelled OmpR (Fl-OmpR) with EnvZc was determined to be 1.96 +/- 0.28 micro M. We demonstrated that OmpR-P as well as OmpR inhibited the interaction of Fl-OmpR with EnvZc. Their 50% inhibitory concentrations were 1.09 +/- 0.25 micro M and 0.89 +/- 0.14 micro M, respectively, under the conditions used. The interaction between His-10-OmpR and EnvZc was also inhibited almost equally with OmpR-P and OmpR. Fluorescein labelling of OmpR was highly heterogeneous as detected by mass spectrometry, even though it slightly affected the OmpR phosphorylation (kinase) and the dephosphorylation of OmpR-P (phosphatase), indicating that EnvZc is able to interact with Fl-OmpR or Fl-OmpR-P as well as with OmpR or OmpR-P as a substrate. We demonstrated that OmpR-P is able to interact with EnvZc with a similar affinity to OmpR and serves as an effective substrate for the EnvZ phosphatase. These findings support the hypothesis that osmotic signals regulate the level of the cellular concentration of OmpR-P by modulating the ratio of kinase to phosphatase activity of the bifunctional enzymatic activities of EnvZ.
Mol Microbiol 2002 Dec
PMID:Interaction of EnvZ, a sensory histidine kinase, with phosphorylated OmpR, the cognate response regulator. 1245 15

The degree of dependence of a lipid bilayer's surface properties on its conformational state is still an unresolved question. Surface properties are functions of molecular organization in the complex interfacial region. In the past, they were frequently measured using fluorescence spectroscopy. Since a fluorescent probe provides information on its local environment, there is a need to estimate the effect caused by the probe itself. In this paper, we address this question by calculating how lipid head-group orientation effects the fluorescence intensity of Fluorescein-PE (a probe that is sensitive to surface potential). In the theoretical model assumed the lipid bilayer state and the interactions between the charged fluorescent probe and the surrounding lipid molecules was evaluated. The results of this theoretical analysis were compared with experimentally obtained data. A lipid bilayer formed from DPPC was chosen as the experimental system, since it exhibits all the major conformational states within a narrow temperature range of 30 degrees C-45 degrees C. Fluorescein-PE fluorescence intensity depends on local pH, which in turn is sensitive to local electrostatic potential in the probe's vicinity. This local electrostatic potential is generated by lipid head-group dipole orientation. We have shown that the effect of the probe on lipid bilayer properties is limited when the lipid bilayer is in the gel phase, whereas it is more pronounced when the membrane is liquid-crystalline. This implies that Fluorescein-PE is a good reporter of local electrostatic fields when the lipid bilayer is in the gel phase, and is a poor reporter when the membrane is in the liquid-crystalline state.
Cell Mol Biol Lett 2003
PMID:The dependence of Fluorescein-PE fluorescence intensity on lipid bilayer state. Evaluating the interaction between the probe and lipid molecules. 1466 17

Germination is a process which characterized with nescient synthesis of genes. Among the genes synthesized during the germination of wheat embryos, germin genes, proteins and their enzymatic activity were defined. Germin is a water soluble homopentameric glycoprotein which is remarkable resistant to degradation by a broad range of proteases including pepsin. Germin proteins found to have strong oxalate oxidase activity which produces hydrogen peroxide by degrading oxalic acid. The current study, aimed to localize the germin genes, proteins and enzymatic activities in developing coleoptiles which is a rapidly growing protective tissue of leaf primordium and shoot apex. Non-radioactively labeled germin riboprobes were employed to localize germin mRNAs in situ. FITC (Fluorescein isothiocyanate) and alkaline phosphatase linked anti-germin antibodies were used to localize germin proteins under the fluorescence and light microscopy and finally germin enzymatic activity was localized by using appropriate enzyme assay. The results revealed that in coleoptiles germin genes, proteins and their enzymatic activity were predominantly associated with the cells of epidermis and vascular bundle sheath cells.
J Biochem Mol Biol 2004 May 31
PMID:Localization of germin genes and their products in developing wheat coleoptiles. 1546 16

Diagnostic anatomic pathologists play an important role in the care of patients through their careful evaluation of morphological features in routinely prepared histological sections stained with Hematoxylin and Eosin. Morphological assessment of tissue sections, backed by over one hundred years of experience is powerful and allows for the accurate classification and diagnosis of the majority of disease states within pathologically altered tissues. However, the appearance of cells and their architectural arrangement within a morphologically complex tissue represents only a fraction of the information, which is contained within a histological section. These tissues also contain all of the cellular proteins and expressed genes, which help to ultimately determine the biological behavior of cells, as well as provide clues to the origins and pathogenesis of disease states. Technical and theoretical advances in our understanding of cellular biology have provided pathologists with powerful tools to probe beyond pure morphology into the abnormalities in both protein and gene expression that underlie human disease. These tools, which include immunohistochemistry and in situ hybridization, are playing an increasingly important role in diagnostic pathology, as well as in translational research. This review will focus on the emerging role of in situ hybridization within clinical and research laboratories, and will highlight a number of technical advances that have expanded the application of this technology.
J Mol Histol 2004 Aug
PMID:In situ hybridization in the pathology laboratory: general principles, automation, and emerging research applications for tissue-based studies of gene expression. 1561 13

The enzyme-linked immunospot (ELISPOT) assay was originally developed for the detection of individual antibody secreting B-cells. Since then, the method has been improved, and ELISPOT is used for the determination of the production of tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, or various interleukins (IL)-4, IL-5. ELISPOT measurements are performed in 96-well plates with nitrocellulose membranes either visually or by means of image analysis. Image analysis offers various procedures to overcome variable background intensity problems and separate true from false spots. ELISPOT readers offer a complete solution for precise and automatic evaluation of ELISPOT assays. Number, size, and intensity of each single spot can be determined, printed, or saved for further statistical evaluation. Cytokine spots are always round, but because of floating edges with the background, they have a nonsmooth borderline. Resolution is a key feature for a precise detection of ELISPOT. In standard applications shape and edge steepness are essential parameters in addition to size and color for an accurate spot recognition. These parameters need a minimum spot diameter of 6 pixels. Collecting one single image per well with a standard color camera with 750 x 560 pixels will result in a resolution much too low to get all of the spots in a specimen. IFN-gamma spots may have only 25 microm diameters, and TNF-alpha spots just 15 microm. A 750 x 560 pixel image of a 6-mm well has a pixel size of 12 microm, resulting in only 1 or 2 pixel for a spot. Using a precise microscope optic in combination with a high resolution (1300 x 1030 pixel) integrating digital color camera, and at least 2 x 2 images per well will result in a pixel size of 2.5 microm and, as a minimum, 6 pixel diameter per spot. New approaches try to detect two cytokines per cell at the same time (i.e., IFN-gamma and IL-5). Standard staining procedures produce brownish spots (horseradish peroxidase) and blue spots (alkaline phosphatase). Problems may occur with color overlaps from cells producing both cytokines, resulting in violet spots. The latest experiments therefore try to use fluorescence labels as a marker. Fluorescein isothiocyanate results in green spots and Rhodamine in red spots. Cells producing both cytokines appear yellow. These colors can be separated much easier than the violet, red, and blue, especially using a high resolution.
Methods Mol Biol 2005
PMID:High resolution as a key feature to perform accurate ELISPOT measurements using Zeiss KS ELISPOT readers. 1593 49

Speckles are nuclear bodies that contain pre-mRNA splicing factors and polyadenylated RNA. Because nuclear poly(A) RNA consists of both mRNA transcripts and nucleus-restricted RNAs, we tested whether poly(A) RNA in speckles is dynamic or rather an immobile, perhaps structural, component. Fluorescein-labeled oligo(dT) was introduced into HeLa cells stably expressing a red fluorescent protein chimera of the splicing factor SC35 and allowed to hybridize. Fluorescence correlation spectroscopy (FCS) showed that the mobility of the tagged poly(A) RNA was virtually identical in both speckles and at random nucleoplasmic sites. This same result was observed in photoactivation-tracking studies in which caged fluorescein-labeled oligo(dT) was used as hybridization probe, and the rate of movement away from either a speckle or nucleoplasmic site was monitored using digital imaging microscopy after photoactivation. Furthermore, the tagged poly(A) RNA was observed to rapidly distribute throughout the entire nucleoplasm and other speckles, regardless of whether the tracking observations were initiated in a speckle or the nucleoplasm. Finally, in both FCS and photoactivation-tracking studies, a temperature reduction from 37 to 22 degrees C had no discernible effect on the behavior of poly(A) RNA in either speckles or the nucleoplasm, strongly suggesting that its movement in and out of speckles does not require metabolic energy.
Mol Biol Cell 2006 Mar
PMID:Rapid, diffusional shuttling of poly(A) RNA between nuclear speckles and the nucleoplasm. 1637 3

This study demonstrates that the single mitochondrion of the sea urchin sperm undergoes a shape change at fertilization that is linked to respiration. The mitochondrion swells and shifts to the lateral side of the sperm head on contact with the homologous egg jelly or egg surface; Mg(2+)- or Na(+)-free seawater or respiratory inhibitors also induce this change. During the mitochondrial deformation, the sperm decreases the rate of oxygen consumption and their redox-state of cytochromes is disrupted b-c(1)/c. Simultaneously, the adenine nucleotides content changes precipitously. This suggests that mitochondrial morphology is strongly associated with respiratory activities in the sea urchin sperm. These changes in mitochondrial morphology and function are similar to the mitochondrial changes in apoptotic cells such as swelling, decrease in its membrane potential, and release of cytochrome c. In apoptotic cells, the exposure of phosphatidylserine from the inner to outer leaflet of the plasma membrane is one of prominence phenomena. This change was visualized by staining the sea urchin sperm with Annexin V-Fluorescein. It is possible that mitochondrial deformation is an initial sign of sperm destruction, which like as apoptotic cells.
Mol Reprod Dev 2006 Oct
PMID:Fertilization induced changes in sea urchin sperm: mitochondrial deformation and phosphatidylserine exposure. 1686 19

Apoptosis, an active process of cell self-destruction, is associated with myocardial ischemia. The redistribution of phosphatidylserine (PS) from the inner to the outer leaflet of the cell membrane is an early event in apoptosis. Annexin V, a protein with high specificity and tight binding to PS, was used to identify and localize apoptosis in the ischemic heart.Fluorescein-labeled annexin V has been used routinely for the assessment of apoptosis in vitro. For the detection of apoptosis in vivo, positron emission tomography and single-photon emission computed tomography have been shown to be suitable tools. In view of the relatively low spatial resolution of nuclear imaging techniques, we developed a high-resolution contrast-enhanced magnetic resonance imaging (MRI) method that allows rapid and noninvasive monitoring of apoptosis in intact organs. Instead of employing superparamagnetic iron oxide particles linked to annexin V, a new T1 contrast agent was used. To this effect, annexin V was linked to gadolinium diethylenetriamine pentaacetate (Gd-DTPA)-coated liposomes. The left coronary artery of perfused isolated rat hearts was ligated for 30 min followed by reperfusion. T(1) and T(2)* images were acquired by using an 11.7-T magnet before and after intracoronary injection of Gd-DTP-labeled annexin V to visualize apoptotic cells. A significant increase in signal intensity was visible in those regions containing cardiomyocytes in the early stage of apoptosis. Because labeling of early apoptotic cell death in intact organs by histological and immunohistochemical methods remains challenging, the use of Gd-DTPA-labeled annexin V in MRI is clearly an improvement in rapid targeting of apoptotic cells in the ischemic and reperfused myocardium.
Mol Imaging
PMID:Assessment of cardiovascular apoptosis in the isolated rat heart by magnetic resonance molecular imaging. 1695 25

The ability to image biochemical and phenotypical changes in living cells has become crucial for the investigation and understanding of the molecular mechanisms that govern all physiological cellular functions in health and disease. Genetically encoded reporters derived from fluorescent proteins (FPs) have proved to be extremely useful for localization and interaction studies in living cells. However, the large size and spectral properties of FP impose certain limitations for their use. The recently developed Fluorescein Arsenical Hairpin (FlAsH/tetracysteine) binder technology emerged as a promising alternative to FP for protein labeling and cellular localization studies. The combination of a small genetically encoded peptide tag with a small molecule detection reagent makes this technology particularly suitable for the investigation of biochemical changes in living cells that are difficult to approach with fluorescent proteins as molecular tags. We describe the practical application of this technology to image protein dynamics in living cells.
Methods Mol Biol 2007
PMID:Protein labeling with FlAsH and ReAsH. 1698 5


<< Previous 1 2 3 4 5 6 7 8 9 Next >>