Exclusive Labeling Research Articles

Hydrogen Bonding in Alzheimer’s Amyloid‐β Fibrils Probed by 15N{17O} REAPDOR Solid‐State NMR Spectroscopy

An exclusive label: 15N{17O} REAPDOR NMR was used to validate intermolecular C17O⋅⋅⋅H15N hydrogen bonding in Ac-Aβ(16–22)-NH2 (see scheme) and Aβ(11–25) amyloid fibrils, which are associated with Alzheimer's disease, by selectively labeling them with 17O and 15N. This method was effective for confirming the structure of these fibrils, and could be useful for a number of other biological samples.

Mostly Heterosexual and Mostly Gay/Lesbian: Evidence for New Sexual Orientation Identities

A sample of 1,784 individuals responded to an online survey advertised on the Facebook social networking website. We explored the sexual orientation continuum by focusing on three components: self-reported sexual orientation identity, sexual attraction, and sexual partners. Results supported a 5-category classification of identity (heterosexual, mostly heterosexual, bisexual, mostly gay/lesbian, gay/lesbian) in that two added identity labels (mostly heterosexual and mostly gay/lesbian) were frequently chosen by participants and/or showed unique patterns of attraction and partners, distinct from their adjacent identities (heterosexual and bisexual, and bisexual and gay/lesbian, respectively). Those who reported an exclusive label (heterosexual, gay/lesbian) were not necessarily exclusive in other components; a significant minority of heterosexuals and the majority of gays/lesbians reported some attraction and/or partners toward their nonpreferred sex. The five identity groups differed in attraction and partners in a manner consistent with a continuous, rather than a categorical, distribution of sexual orientation. Findings also supported a sexual orientation continuum as consisting of two, rather than one, distinct dimensions (same- and other-sex sexuality). Having more same-sex sexuality did not necessarily imply having less other-sex sexuality, and vice versa. More men than women were at the exclusive ends of the continuum; however, men were not bimodally distributed in that a significant minority reported nonexclusivity in their sexuality.

Continental Drift: The Disjunction of North and Sub-Saharan Africa

Research and popular imaginative views of in the last few decades have tended to leave out the northern region, even when referring to the continent as a whole. In many academic disciplines, Africa and The Arab World are mutually exclusive labels, and separating between North and sub-Saharan has become accepted to such an extent that it has shaped our perception of African Studies as a field. How have literatures of the two regions come to be separated and so rarely studied together despite strong links caused by geographic proximity? Why have scholars of African literatures focused largely on sub-Saharan Africa? To begin answering these questions, it is necessary not only to address literary mat- ters—including issues such as the impact of the publishing industry in codifying a canon of African literature, but also to consider factors relating to the political climate of the twentieth century.

Continental Drift: The Disjunction of North and Sub-Saharan Africa

Research and popular imaginative views of Africa in the last few decades have tended to leave out the northern region, even when referring to the continent as a whole. In many academic disciplines, "Africa" and "The Arab World" are mutually exclusive labels, and separating between North and sub-Saharan Africa has become accepted to such an extent that it has shaped our perception of African Studies as a field. How have literatures of the two regions come to be separated and so rarely studied together despite strong links caused by geographic proximity? Why have scholars of African literatures focused largely on sub-Saharan Africa? To begin answering these questions, it is necessary not only to address literary mat- ters—including issues such as the impact of the publishing industry in codifying a canon of African literature, but also to consider factors relating to the political climate of the twentieth century.

Ultrasensitive in situ visualization of active glucocerebrosidase molecules

Deficiency of glucocerebrosidase (GBA) underlies Gaucher disease, a common lysosomal storage disorder. Carriership for Gaucher disease has recently been identified as major risk for parkinsonism. Presently, no method exists to visualize active GBA molecules in situ. We here report the design, synthesis and application of two fluorescent activity-based probes allowing highly specific labeling of active GBA molecules in vitro and in cultured cells and mice in vivo. Detection of in vitro labeled recombinant GBA on slab gels after electrophoresis is in the low attomolar range. Using cell or tissue lysates, we obtained exclusive labeling of GBA molecules. We present evidence from fluorescence-activated cell sorting analysis, fluorescence microscopy and pulse-chase experiments of highly efficient labeling of GBA molecules in intact cells as well as tissues of mice. In addition, we illustrate the use of the fluorescent probes to study inhibitors and tentative chaperones in living cells.

Synthesis, Properties, and Applications of Diazotrifluropropanoyl‐Containing Photoactive Analogs of Farnesyl Diphosphate Containing Modified Linkages for Enhanced Stability

Photoactive analogs of farnesyl diphosphate (FPP) are useful probes in studies of enzymes that employ this molecule as a substrate. Here, we describe the preparation and properties of two new FPP analogs that contain diazotrifluoropropanoyl photophores linked to geranyl diphosphate via amide or ester linkages. The amide-linked analog (3) was synthesized in 32P-labeled form from geraniol in seven steps. Experiments with Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) showed that 3 is an alternative substrate for the enzyme. Photolysis experiments with [(32)P]3 demonstrate that this compound labels the beta-subunits of both farnesyltransferase and geranylgeranyltransferase (types 1 and 2). However, the amide-linked probe 3 undergoes a rearrangement to a photochemically unreactive isomeric triazolone upon long term storage making it inconvenient to use. To address this stability issue, the ester-linked analog 4 was prepared in six steps from geraniol. Computational analysis and X-ray crystallographic studies suggest that 4 binds to protein farnesyl transferase (PFTase) in a similar fashion as FPP. Compound 4 is also an alternative substrate for PFTase, and a 32P-labeled form selectively photocrosslinks the beta-subunit of ScPFTase as well as E. coli farnesyldiphosphate synthase and a germacrene-producing sesquiterpene synthase from Nostoc sp. strain PCC7120 (a cyanobacterial source). Finally, nearly exclusive labeling of ScPFTase in crude E. coli extract was observed, suggesting that [32P]4 manifests significant selectivity and should hence be useful for identifying novel FPP-utilizing enzymes in crude protein preparations.

Some results on exclusive sum graphs

Let N(Z) denote the set of all positive integers (integers). The sum graph G+(S) of a finite subset S⊂N(Z) is the graph (S,E) with uv∈E if and only if u+v∈S. A graph G is said to be an (integral) sum graph if it is isomorphic to the sum graph of some S⊂N(Z). A sum labelling S is called an exclusive sum labelling if u+v∈S∖V(G) for any edge uv∈E(G). We say that G is labeled exclusively. The least number r of isolated vertices such that G∪rK1 is an exclusive sum graph is called the exclusive sum number e(G) of graph G. In this paper, we discuss the exclusive sum number of disjoint union of two graphs and the exclusive sum number of some graph classes.

Exclusive Sum Labeling of Graphs: A Survey

All sum graphs are disconnected. In order for a connected graph to bear a sum labeling, the graph is considered in conjunction with a number of isolated vertices, the labels of which complete the sum labeling for the disjoint union. The smallest number of isolated vertices that must be added to a graph H to achieve a sum graph is called the sum number of H; it is denoted by σ(H). A sum labeling which realizes as a sum graph is called an optimal sum labeling of H.In this paper we survey a new type of labeling based on summation, the exclusive sum, labeling. A sum labeling L is called exclusive sum labeling with respect to a subgraph H of G if L is a sum labeling of G where H contains no working vertex. The exclusive sum number ∊(H) of a graph H is the smallest number r such that there exists an exclusive sum labeling L which realizes as a sum graph. A labeling L is an optimal exclusive sum labeling of a graph H if L is a sum labeling of H ≼ K∊(H) and H contains no working vertex.

Nucleotide-Binding Sites of the Heterodimeric LmrCD ABC-Multidrug Transporter of Lactococcus lactis Are Asymmetric

LmrCD is a lactococcal, heterodimeric multidrug transporter, which belongs to the ABC superfamily. It consists of two half-transporters, LmrC and LmrD, that are necessary and sufficient for drug extrusion and ATP hydrolysis. LmrCD is asymmetric in terms of the conservation of the functional motifs of the nucleotide-binding domains (NBDs). Important residues of the nucleotide-binding site of LmrC and the C loop of LmrD are not conserved. To investigate the functional importance of the LmrC and LmrD subunits, the putative catalytic base residue adjacent to the Walker B motif of both NBDs were substituted for the respective carboxamides. Our data demonstrate that Glu587 of LmrD is essential for both drug transport and ATPase activity of the LmrCD heterodimer, whereas mutation of Asp495 of LmrC has a less severe effect on the activity of the complex. Structural and/or functional asymmetry is further demonstrated by differential labeling of both subunits by 8-azido-[alpha-32P]ATP, which, at 4 degrees C, occurs predominantly at LmrC, while aluminiumfluoride (AlF(x))-induced trapping of the hydrolyzed nucleotide at 30 degrees C results in an almost exclusive labeling of LmrD. It is concluded that the LmrCD heterodimer contains two structurally and functionally distinct NBDs.

Novel Assay Utilizing Fluorochrome-Tagged Physostigmine (Ph-F) to In Situ Detect Active Acetylcholinesterase (AChE) Induced during Apoptosis

It was recently reported that acetylcholinesterase (AChE) is expressed in cells undergoing apoptosis and that its presence is essential for assembly of the apoptosome and subsequent caspase-9 activation. To obtain a marker of active AChE that could assay this enzyme in live intact cells and be applicable to fluorescence microscopy and cytometry, the fluorescein-tagged physostigmine (Ph-F), high affinity ligand (inhibitor) reactive with the active center of AChE, was constructed and tested for its ability to in situ label AChE and measure its induction during apoptosis. Ph-F inhibited cholinesterase activity in vitro (IC50 = 10-6 and 5x10-6 M for equine butyrycholinesterase and human erythrocyte AChE, respectively) and was a selective marker of cells and structures that were AChE-positive. Thus, exposure of mouse bone marrow cells to Ph-F resulted in the exclusive labeling of megakaryocytes, and of the diaphragm muscle, preferential labeling of the nerve-muscle junctions (end-plates). During apoptosis of carcinoma HeLa cells and leukemic HL-60 or Jurkat cells triggered either by the DNA topoisomerase 1 inhibitor topotecan (TPT) or by oxidative stress (H2O2), the cells become reactive with Ph-F. Their Ph-F derived fluorescence was measured by flow and laser scanning cytometry. The appearance of Ph-F binding sites during apoptosis was preceded by the loss of mitochondrial potential, was concurrent with the presence of activated caspases, and was followed by loss of membrane integrity. At a very early stage of apoptosis, when nucleolar segregation was apparent, the Ph-F binding sites were distinctly localized within the nucleolus and at later stages of apoptosis in the cytoplasm. During apoptosis triggered by TPT, Ph-F binding was preferentially induced in S-phase cells. Our data on megakarocytes and end-plates indicate that Ph-F reacts with active sites of AChE, and can be used to reveal the presence of this enzyme in live cells and possibly to study its expression in disorders of the neurological cholinergic system. The findings are also compatible with the reports that AChE may be induced during apoptosis. In fact, the simple and rapid Ph-F binding assay may serve as a convenient marker of apoptotic cells. However, the proposed role of active AChE as an essential factor for assembly of the apoptosome and caspase activation is arguable because the AChE inhibitors Ph, Ph-F and BW284c51did not protect the cells from apoptosis induced by TPT or H2O2. Further studies are thus needed to ascertain the induction and role of AChE in apoptosis.

UT-A3: localization and characterization of an additional urea transporter isoform in the IMCD.

UT-A3 has recently been identified as a splicing variant transcript of the UT-A gene present in the kidney. To study the cellular and subcellular localization of UT-A3, we raised a new polyclonal antibody to its COOH-terminal end. Immunoblots identified bands at 44 and 67 kDa predominately in the inner medulla and showed that the antibody does not recognize UT-A1. Deglycosylation with PNGase decreased the molecular mass of both forms to 40 kDa. UT-A3 is most abundant in the inner third of the inner medulla and is present in membrane fractions. Cell fractionation studies showed that UT-A3 is only detectable in inner medullary collecting duct (IMCD) cells. These observations were confirmed with immunolocalization studies demonstrating an exclusive labeling of IMCD cells. Double-labeling studies with anti-Na-K-ATPase demonstrated UT-A3 in intracellular membranes and in the apical region but were incompatible with a basolateral site for UT-A3. Although the function of this isoform in the inner medulla is unknown, the large abundance suggests that it may be important in the renal handling of urea.

Object label learning by middle- and working-class, black and white, younger and older preschool children

The present study examined the ability of younger and older preschool children from different backgrounds (16 middle-class black, 16 middle-class white, 16 working-class black, and 16 working-class white) to map labels to objects and to establish inclusion relationships. The children were taught novel labels for perceptually related and unrelated unfamiliar objects. Although the groups were similar in their ability to comprehend the first label during the first session, white children produced the first label more. When additional labels were taught during subsequent sessions, the differences among the groups of children were augmented. Although children from all backgrounds applied labels to objects based on initial labelings, middle-class, white, and older children did this to a greater extent than working-class, black, and younger children. Evidence for the use of inclusion relations for perceptually related target objects was not found for the children; rather, the children used a mutually exclusive labeling strategy.

Structural and Immunological Characterization of the Cuscuta pentagona L. Chloroplast

Structural and immunochemical studies were used to determine the photosynthetic potential of the dodder (Cuscuta pentagona) chloroplast. Ultrastructural studies revealed that thylakoid membranes of pre-parasitic phase Cuscuta pentagona are almost all organized into long, overlapping grana stacks of mainly two to five thylakoids with little space between adjacent stacks. Immunoblots reveal chloroplast proteins associated with PSI and 11, as well as cytochrome f and plastocyanin. Stromal extracts contained immmunologically-detectable RuBisCO and phosphoribulokinase. Cytochemical localizations of the oxidizing side of PSI showed product localization on the lumen side of the thylakoid. Immunocytochemical localizations of RuBisCO reveal exclusive labeling in the stroma, whereas antibodies to the PSII proteins, light-harvesting Chl a/b complex and the oxygen-evolving complex of PSII, are concentrated over the thylakoids. A limited capacity for C02 fixation was found in seedlings by monitoring exchange rates in the presence and absence of atrazine. These data indicate that the chloroplast from this species of dodder contains a number of the proteins required for a successful fixation of C02 and the proteins in the thylakoids are organized much like other higher plants, with the exception of the large percentage of the thylakoids organized into grana.

Irreversible Inhibition of Lysyl Oxidase by Homocysteine Thiolactone and Its Selenium and Oxygen Analogues: IMPLICATIONS FOR HOMOCYSTINURIA

Homocysteine thiolactone, selenohomocysteine lactone, and homoserine lactone were found to be competitive, irreversible inhibitors of lysyl oxidase, with KI values of 21 +/- 3 microM, 8.3 +/- 2.2 microM, and 420 +/- 56 microM, respectively. The first order rate constants for inactivation (k2) of the enzyme varied over a much smaller range, ranging from 0.12 to 0.18 to 0.28 min-1 for the Se-, thio-, and O-lactones, respectively. Mutually exclusive labeling of the enzyme by [1-14C]beta-aminopropionitrile, [U-14C]phenylhydrazine, or [35S]homocysteine thiolactone was observed. These labeling results, together with the closely similar perturbations of the near UV-visible spectra of lysyl oxidase and of a model of its lysine tyrosylquinone cofactor by the thiolactone, indicate that the lactones likely derivatize and reduce the active site carbonyl cofactor. Substitution with deuterium at the alpha-carbon of the thiolactone caused a deuterium kinetic isotope effect on k2 of 3.2 +/- 0.2, consistent with the involvement of rate-limiting alpha-proton abstraction during lactone-induced inactivation of the enzyme. The activities of plasma amine oxidase and diamine oxidase were only minimally reduced at concentrations of the sulfur or selenium lactones that fully inhibited lysyl oxidase. Thus, these lactones constitute a new category of mechanism-based inactivators selective for lysyl oxidase. Further, these results may relate to the development of connective tissue defects seen in homocystinuria.

The bicoid-related homeoprotein Ptx1 defines the most anterior domain of the embryo and differentiates posterior from anterior lateral mesoderm.

Ptx1 is a member of the small bicoid family of homeobox-containing genes; it was isolated as a tissue-restricted transcription factor of the pro-opiomelanocortin gene. Its expression during mouse and chick embryogenesis was determined by in situ hybridization in order to delineate its putative role in development. In the head, Ptx1 expression is first detected in the ectoderm-derived stomodeal epithelium at E8.0. Initially, expression is only present in the stomodeum and in a few cells of the rostroventral foregut endoderm. A day later, Ptx1 mRNA is detected in the epithelium and in a streak of mesenchyme of the first branchial arch, but not in other arches. Ptx1 expression is maintained in all derivatives of these structures, including the epithelia of the tongue, palate, teeth and olfactory system, and in Rathke's pouch. Expression of Ptx1 in craniofacial structures is strikingly complementary to the pattern of goosecoid expression. In addition, Ptx1 is expressed early (E6.8) in posterior and extraembryonic mesoderm, and in structures that derive from these. The restriction of expression to the posterior lateral plate is later evidenced by exclusive labelling of the hindlimb but not forelimb mesenchyme. In the anterior domain of expression, the stomodeum was shown by fate mapping to derive from the anterior neural ridge (ANR) which represents the most anterior domain of the embryo. The concordance between these fate maps and the stomodeal pattern of Ptx1 expression supports the hypothesis that Ptx1 defines a stomodeal ectomere, which lies anteriorly to the neuromeres that have been suggested to constitute units of a segmented plan directing head formation.

Conformational changes and fusion activity of vesicular stomatitis virus glycoprotein: [125I]iodonaphthyl azide photolabeling studies in biological membranes.

The interaction of VSV glycoprotein (VSV G) with biological membranes was studied by photosensitized labeling. The method is based on photosensitized activation by the fluorescent lipid analog 3,3'-dioctadecyloxacarbocyanine (DiO) of a hydrophobic probe, [125I]iodonaphthyl azide (125INA), that rapidly partitions into the membrane bilayer of virus and cells. 125INA labeling of proteins and lipids can be confined to the site of chromophore localization by photosensitized labeling. Photoactivation using visible light of target membrane labeled with DiO and 125INA, to which unlabeled virions are bound, results in exclusive labeling of envelope glycoproteins inserted into the target membrane [Pak et al. (1994) J. Biol. Chem. 269, 14614]. In this study, we labeled lipid symmetric erythrocyte ghosts with 125INA and DiO. Photosensitized activation of VSV prebound to labeled ghosts with visible light resulted in VSV G labeling under fusogenic conditions. Photoactivation of 125INA by UV light, which is nonspecific, produced labeled VSV G at both acidic and neutral pH. Photosensitized labeling of VSV G by DiO-125INA-ghosts was also observed at pH 5.5, 4 degrees C, in the absence of mixing between viral and cellular lipids, suggesting insertion of the ectodomain of VSV G. Soluble VSV G lacking the transmembrane domain inserted into DiO-125INA-ghosts under the same conditions as intact VSV G. DiO inserted into intact VSV appeared to be a suitable fluorophore for continuous kinetic measurements of membrane fusion by fluorescence dequenching. Our photosensitized labeling results establish biochemical correlates for the three states of VSV G, which we had proposed based on kinetic data [Clague et al., Biochemistry 29, 1303]. In addition, we found that VSV G insertion into the target membrane is reversible, suggesting a "velcro"-like attachment of the fusogenic domain with the target membrane.

Exclusive Labels: Indexing the National “We” in Commemorative and Oppositional Exhibitions

Exclusive Labels: Indexing the National “We” in Commemorative and Oppositional Exhibitions

Dynactin Phosphorylation Is Modulated in Response to Cellular Effectors

Reversible protein phosphorylation has been implicated in the regulation of organelle transport by cytoplasmic dynein. Motor function may be modulated directly by the phosphorylation of dynein or through the phosphorylation of an accessory factor. Dynactin binds to cytoplasmic dynein and is a required activator for dynein-driven vesicular motility. In metabolic labeling studies we have determined that the p150Gluedsubunit of dynactin is a phosphoprotein. Treatment of Rat2 cells with okadaic acid or with activators of protein kinase A or protein kinase C caused a marked increase in the incorporation of32P into p150Glued; the increased phosphorylation correlated with activated vesicular transport. Phosphoamino-acid analysis of p150Gluedisolated from cells treated with okadaic acid or with activators of either protein kinase A or protein kinase C indicated exclusive labeling of phosphoserine. These results suggest that the phosphorylation of dynactin may serve to regulate intracellular transport catalyzed by cytoplasmic dynein.

All-trans-Retinoic Acid Regulates Retinol and 3,4-Didehydroretinol Metabolism in Cultured Human Epidermal Keratinocytes

The potential for all-trans-retinoic acid to regulate the metabolism of 3H-retinol and 3H-3,4-didehydroretinol was examined in cultured human epidermal keratinocytes. Confluent cultures were treated daily with medium containing 5% fetal bovine serum or the same medium supplemented with nanomolar concentrations of all-trans-retinoic acid for up to 3 d. During the last 24 of treatment, cells were incubated with 3H-retinol or 3H-3,4-didehydroretinol for 24 h (isotopic steady state) to label the endogenous retinoids. After the labeling period, one group of cells was harvested and another group was allowed to incubate for an additional 24 h in the absence of medium retinol for the determination of endogenous 3H-retinoid utilization. The 3H-retinoids present in cells were extracted and quantitated by reverse-phased high-pressure liquid chromatography. Keratinocytes treated with retinoic acid and labeled with 3H-retinol exhibited time- and concentration-dependent (i) increases in retinyl ester mass, (ii) increases in the rate of retinyl ester synthesis, (iii) decreases in retinyl ester utilization, and (iv) decreases in the cellular concentrations of retinoic and 3,4-didehydroretinoic acids. There was no effect of exogenous retinoic acid on its own metabolism. Cells labeled with 3H-3,4-didehydroretinol exhibited exclusive labeling of vitamin A2-related retinoids suggesting that the A1 to A2 conversion is not reversible. Treatment of cells with low nanomolar concentrations of retinoic acid decreased the utilization of 3,4-didehydroretinyl esters, decreased the production of 3,4-didehydroretinoic acid but had no effect on the synthesis of 3,4-didehydroretinol or its esters. The results demonstrate that keratinocytes respond to extracellular retinoic acid by decreasing endogenous production of active retinoids, sequestering extracellular substrate retinol as retinyl ester, and decreasing ester utilization.

Glial fibrillary acidic protein in the brain of the caecilian Typhlonectes natans (Amphibia, Gymnophiona): an immunocytochemical study

The astroglia of adult and juvenile (metamorphosed) Typhlonectes natans (Fischer) was investigated immunocytochemically with a monoclonal antibody directed against glial fibrillary acidic protein (GFAP). The astroglia of this member of the Order Gymnophiona of the class Amphibia is mainly composed of radial glial cells. Their somata limit the ventricles. They each give rise to a thick process that extends through the periventricular gray and arborizes within the neuropil. At the subpial surface, endfeet establish the membrana gliae limitans externa. Some extraependymal radial glial cells are immunoreactive, but no mammalian-like astrocytes are visualized. In the spinal cord, perikarya of radial glia are displaced from the GFAP-immunonegative ependyma. Perivascular endfeet and processes lining blood vessels are abundantly labeled. An increase in GFAP immunoreactivity extends from the exclusive labeling of subpial endfeet in newborn, recently metamorphosed animals, to the subsequent staining of distal processes and of the entire cell in older juveniles. The midline glia of the brainstem is immunoreactive at all ages examined. Strong glial wedges separate and delineate fiber tracts. Radial glial fibers in the habenulae are particularly thick and exhibit strong GFAP immunoreactivity, even in juveniles where GFAP immunoreactivity is otherwise minimal. The pattern of GFAP immunolabeling in the caecilian T. natans is similar to that in salamanders, but not to that in frogs.