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Terminus
Kinds of Terminus Selected AbstractsAccelerated iterative strategy for the divergent synthesis of dendritic macromolecules using a combination of living radical polymerization and an irreversible terminator multifunctional initiatorJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 20 2005Virgil Percec Abstract Our laboratory has reported the elaboration of an iterative strategy for the synthesis of dendritic macromolecules from conventional monomers. This synthetic method involves a combination of self-regulated metal-catalyzed living radical polymerization initiated from arenesulfonyl chlorides and an irreversible terminator multifunctional initiator (TERMINI). The previous TERMINI, (1,1-dimethylethyl)[[1-[3,5-bis(S -phenyl-4- N,N, diethylthiocarbamate)phenyl]ethenyl]oxy]dimethylsilane, was prepared in nine reaction steps. The replacement of the previous TERMINI with one that requires only three steps for its synthesis, diethylthiocarbamic acid S -{3-[1-(tert -butyl-dimethyl-silanyloxy)-vinyl]-5-diethylcarbamoylsulfanyl-phenyl} ester, and the use of the more reactive Cu2S/2,2,-bipyridine rather than the Cu2O/2,2,-bipyridine self-regulated catalyst have generated an accelerated method for the synthesis of dendritic macromolecules. This method provides rational design strategies for the synthesis of dendritic macromolecules with different compaction by the use of a single monomer. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 4894,4906, 2005 [source] Towards Control of Dendrimer Properties by Reversible Exchange of Termini: Synthesis and Characterization of Diverse Porphyrin DendrimersISRAEL JOURNAL OF CHEMISTRY, Issue 1 2009Meital Shema-Mizrachi Porphyrin dendrimers with boronic ester, aldehyde, and pyridil termini were synthesized and fully characterized. These dendrimers have the potential to change their physical and chemical properties by reversible alteration of the reactive terminal groups. [source] Synthesis of [2]Rotaxanes by Tritylative Endcapping of in situ Formed Pseudorotaxanes Having Thiol or Hydroxyl Functionality on the Axle Termini.CHEMINFORM, Issue 47 2002Yoshio Furusho No abstract is available for this article. [source] Tailored Control and Optimisation of the Number of Phosphonic Acid Termini on Phosphorus-Containing Dendrimers for the Ex-Vivo Activation of Human MonocytesCHEMISTRY - A EUROPEAN JOURNAL, Issue 16 2008Olivier Rolland Abstract The syntheses of a series of phosphonic acid-capped dendrimers is described. This collection is based on a unique set of dendritic structural parameters,cyclo(triphosphazene) core, benzylhydrazone branches and phosphonic acid surface,and was designed to study the influence of phosphonate (phosphonic acid) surface loading towards the activation of human monocytes ex vivo. Starting from the versatile hexachloro-cyclo(triphosphazene) N3P3Cl6, six first-generation dendrimers were obtained, bearing one to six full branches, that lead to 4, 8, 12, 16, 20 and 24 phosphonate termini, respectively. The surface loading was also explored at the limit of dense packing by means of a first-generation dendrimer having a cyclo(tetraphosphazene) core and bearing 32 termini, and with a first-generation dendrimer based on a AB2/CD5 growing pattern and bearing 60 termini. Human monocyte activation by these dendrimers confirms the requirement of the whole dendritic structure for bioactivity and identifies the dendrimer bearing four branches, thus 16 phosphonate termini, as the most bioactive. [source] IV,Three Moments in the Theory of Definition or Analysis: Its Possibility, Its Aim or Aims, and Its Limit or TerminusPROCEEDINGS OF THE ARISTOTELIAN SOCIETY (HARDBACK), Issue 1pt1 2007David Wiggins The reflections recorded in this paper arise from three moments in the theory of definition and of conceptual analysis. The moments are: (I) Frege's (1894) review of Husserl's Philosophy of Arithmetic (Vol. I), the discussion there of the paradox of analysis, and the division that Frege marks, ensuing upon his distinction of Sinn/sense from Bedeutung/reference, between two different conceptions of definition; (II) Leibniz's still serviceable account (1684, 1704) of a distinction between the clarity and the distinctness of ideas,a distinction that prompts the suggestion that the guiding purpose of lexical definition is Leibnizian clarity whereas that of real definition (as Aristotle has us conceive it) is inseparable from the pursuit of Leibnizian distinctness; (III) Leibniz's speculations (1679) concerning the limit or terminus of analysis. The apparent failure of these speculations, casting doubt as it does upon the aspirations that give rise to them (aspirations not necessarily or entirely alien to the Zeitgeist of our own epoch), points to the long-standing need to reconfigure the philosophical business of enquiry into concepts. [source] The SSU rDNA Coding Region of a Filose Amoeba Contains a Group I Intron Lacking the Universally Conserved G at the 3,-Terminus,THE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 6 2000DEBASHISH BHATTACHARYA ABSTRACT. We sequenced small subunit ribosomal DNA (rDNA) PCR-fragments of sizes 2.3 kb and 2.9 kb isolated from a culture of the red alga, Porphyru spiralis var. spiralis. Phylogenetic analysis of the 2.3-kb fragment showed that it encoded the sequence of a contaminant filose amoeba. The Nuclearia -like amoeba (named strain N-Por) was identified with scanning electron microscopy. Its rDNA sequence was positioned with strong bootstrap support within a diverse protist assemblage that includes filose amoebae, chlor-arachniophytes, cercomonads, and Plasmodiophora brussicae. The rDNA of N-Por contained a group I intron at the conserved 943 position that remarkably, had a U at the 3,-terminus rather than the universally conserved G. [source] Structure,Activity Study on the Spatial Arrangement of the Third Aromatic Ring of Endomorphins 1 and 2 Using an Atypical Constrained C,TerminusCHEMMEDCHEM, Issue 3 2007Ye Yu Dr. Abstract The discovery of endomorphins (EMs) has opened the possibility of searching for new analgesics. However, the design of peptide analgesics has proven to be very difficult as a result of their conformational flexibility and a lack of clarity in structure,activity relationships (SAR). In EMs, the amino acid side chains exhibit considerable conformational flexibility, especially in the third aromatic ring, which is free to adopt a bioactive conformation. To resolve these problems, a series of C,terminus EM analogues, [Xaa4 -R]EMs, modified through the substitution of Phe4 with nonaromatic residues and termination with benzyl groups, were designed to generate conformational constrains of the third aromatic ring by amide bond and torsion angles (,4 and ,4) of Xaa4. Introduction of (S)-,-methyl or (S)/(R)-,-carboxamide on the methylene unit of the benzyl group was designed to produce an atypical topographical constraint (,5) of the third aromatic ring rotation. Interestingly, some EM derivatives, with elimination of the C-terminal carboxamide group and significant changes in the address sequence (Phe4 -NH2), still exhibited higher ,-opioid receptor (MOR) affinity than unmodified EMs. In contrast, some analogues with incorrectly constrained C,termini displayed very low affinity and pharmacological activities. Thus, our results indicate that these EM analogues, with atypical constrained C,termini, provide model compounds with potent MOR agonism. They also give evidence that the proper spatial orientation and conformational restriction of the third aromatic ring are crucial for the interaction of EMs with MOR. [source] Algorithm for Spatial Clustering of Pavement SegmentsCOMPUTER-AIDED CIVIL AND INFRASTRUCTURE ENGINEERING, Issue 2 2009Chientai Yang This article formulates a new spatial search model for determining appropriate pavement preservation project termini. A spatial clustering algorithm using fuzzy c-mean clustering is developed to minimize the rating variation in each cluster (project) of pavement segments while considering minimal project scope (i.e., length) and cost, initial setup cost, and barriers, such as bridges. A case study using the actual roadway and pavement condition data in fiscal year 2005 on Georgia State Route 10 shows that the proposed algorithm can identify more appropriate segment clustering scheme, than the historical project termini. The benefits of using the developed algorithm are summarized, and recommendations for future research are discussed. [source] Expression patterns of focal adhesion associated proteins in the developing retinaDEVELOPMENTAL DYNAMICS, Issue 4 2002Ming Li Abstract Adhesive interactions between integrin receptors and the extracellular matrix (ECM) are intimately involved in regulating development of a variety of tissues within the organism. In the present study, we have investigated the relationships between ,1 integrin receptors and focal adhesion associated proteins during eye development. We used specific antibodies to examine the distribution of ,1 integrin ECM receptors and the cytoplasmic focal adhesion associated proteins, talin, vinculin, and paxillin in the developing Xenopus retina. Immunoblot analysis confirmed antibody specificity and indicated that ,1 integrins, talin, vinculin, and paxillin were expressed in developing retina and in the retinal-derived Xenopus XR1 glial cell line. Triple-labeling immunocytochemistry revealed that talin, vinculin, paxillin, and phosphotyrosine proteins colocalized with ,1 integrins at focal adhesions located at the termini of F-actin filaments in XR1 cells. In the retina, these focal adhesion proteins exhibited developmentally regulated expression patterns during eye morphogenesis. In the embryonic retina, immunoreactivities for focal adhesion proteins were expressed in neuroepithelial cells, and immunoreactivity was especially strong at the interface between the optic vesicle and overlying ectoderm. At later stages, these proteins were expressed throughout all retinal layers with higher levels of expression observed in the plexiform layers, optic fiber layer, and in the region of the inner and outer limiting membrane. Strong immunoreactivities for ,1 integrin, paxillin, and phosphotyrosine were expressed in the radially oriented Müller glial cells at later stages of development. These results suggest that focal adhesion-associated proteins are involved in integrin-mediated adhesion and signaling and are likely to be essential in regulating retinal morphogenesis. © 2002 Wiley-Liss, Inc. [source] Mitochondrial clustering at the vertebrate neuromuscular junction during presynaptic differentiationDEVELOPMENTAL NEUROBIOLOGY, Issue 6 2006Chi Wai Lee Abstract During vertebrate neuromuscular junction (NMJ) development, presynaptic motor axons differentiate into nerve termini enriched in synaptic vesicles (SVs). At the nerve terminal, mitochondria are also concentrated, but how mitochondria become localized at these specialized domains is poorly understood. This process was studied in cultured Xenopus spinal neurons with mitochondrion-specific probe MitoTracker and SV markers. In nerve-muscle cocultures, mitochondria were concentrated stably at sites where neurites and muscle cells formed NMJs, and mitochondria coclustered with SVs where neurites were focally stimulated by beads coated with growth factors. Labeling with a mitochondrial membrane potential-dependent probe JC-1 revealed that these synaptic mitochondria were with higher membrane potential than the extrasynaptic ones. At early stages of bead-stimulation, actin-based protrusions and microtubule fragmentation were observed in neurites at bead contact sites, suggesting the involvement of cytoskeletal dynamics and rearrangement during presynaptic differentiation. Treating the cultures with an actin polymerization blocker, latrunculin A (Ltn A), almost completely abolished the formation of actin-based protrusions and partially inhibited bead-induced mitochondrial and SV clustering, whereas the microtubule disrupting agent nocodazole was ineffective in inhibiting the clustering of mitochondria and SVs. Lastly, in contrast to Ltn A, which blocked bead-induced clustering of both mitochondria and SVs, the ser/thr phosphatase inhibitor okadaic acid inhibited SV clustering but not mitochondrial clustering. These results suggest that at developing NMJs, synaptogenic stimuli induce the clustering of mitochondria together with SVs at presynaptic terminals in an actin cytoskeleton-dependent manner and involving different intracellular signaling molecules. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006 [source] Localization of KCNC1 (Kv3.1) potassium channel subunits in the avian auditory nucleus magnocellularis and nucleus laminaris during developmentDEVELOPMENTAL NEUROBIOLOGY, Issue 2 2003Suchitra Parameshwaran-Iyer Abstract The KCNC1 (previously Kv3.1) potassium channel, a delayed rectifier with a high threshold of activation, is highly expressed in the time coding nuclei of the adult chicken and barn owl auditory brainstem. The proposed role of KCNC1 currents in auditory neurons is to reduce the width of the action potential and enable neurons to transmit high frequency temporal information with little jitter. Because developmental changes in potassium currents are critical for the maturation of the shape of the action potential, we used immunohistochemical methods to examine the developmental expression of KCNC1 subunits in the avian auditory brainstem. The KCNC1 gene gives rise to two splice variants, a longer KCNC1b and a shorter KCNC1a that differ at the carboxy termini. Two antibodies were used: an antibody to the N-terminus that does not distinguish between KCNC1a and b isoforms, denoted as panKCNC1, and another antibody that specifically recognizes the C terminus of KCNC1b. A comparison of the staining patterns observed with the panKCNC1 and the KCNC1b specific antibodies suggests that KCNC1a and KCNC1b splice variants are differentially regulated during development. Although panKCNC1 immunoreactivity is observed from the earliest time examined in the chicken (E10), a subcellular redistribution of the immunoproduct was apparent over the course of development. KCNC1b specific staining has a late onset with immunostaining first appearing in the regions that map high frequencies in nucleus magnocellularis (NM) and nucleus laminaris (NL). The expression of KCNC1b protein begins around E14 in the chicken and after E21 in the barn owl, relatively late during ontogeny and at the time that synaptic connections mature morphologically and functionally. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 165,178, 2003 [source] Electrochemical Aptasensor for the Determination of Cocaine Incorporating Gold Nanoparticles ModificationELECTROANALYSIS, Issue 13 2008Xiaoxia Li Abstract A novel electrochemical aptasensor incorporating a signal enhancement for the determination of cocaine was designed. Gold nanoparticles were self-assembled onto the surface of a gold electrode through 1,6-hexanedithiol. A bifunctional derivative of the 32-base cocaine-binding aptamer with a redox-active ferrocene moiety and a thiol linker group at the termini of the strand was self-assembled onto the surface of gold nanoparticles. The oxidation peak current is linearly related to the concentration of cocaine from 1.0 to 15.0,,M with a detection limit of 0.5,,M. It was found that the sensitivity of the aptasensor with gold nanoparticles modification was ca. 10-fold higher than that of the aptasensor without gold nanoparticles modification. This work demonstrates that gold nanoparticles-assembled gold electrode provides a promising platform for immobilizing aptamer and enhancing the sensitivity. [source] Selective Analysis of Secondary Amines Using Liquid Chromatography with Electrochemical Detection (LC-EC)ELECTROANALYSIS, Issue 21 2006Celia Abstract In a mixture of primary and secondary aliphatic amines, the primary amines were derivatized (masked) with o -phthalaldehyde (OPA) followed by derivatization of the remaining secondary amines with ferrocenecarboxylic acid chloride (FAC). The "tagged" amines were analyzed by LC-EC (liquid chromatography with electrochemical detection) using in-series dual electrode detection. Chemically-reversible oxidation of the FAC tagged secondary amines and their subsequent complementary oxidation and reduction signals coupled with chemically-irreversible oxidation of OPA tagged primary amines provided the selectivity for quantitative secondary amine analysis. The procedure was also applied for the selective identification of fragment 4,11 (N -terminus-proline) of Substance P in the presence of other Substance P fragments with primary amino acids as their N -termini. [source] N-terminal destruction signals lead to rapid degradation of the major histocompatibility complex class II transactivator CIITAEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 8 2003Felix Schnappauf Abstract Major histocompatibility complex (MHC) class II molecules play an essential role for the cellular immune response by presenting peptide antigens to CD4+ T cells. MHC class II molecules and genes show a highly complex expression pattern, which is orchestrated through a master regulatory factor, called CIITA (class II transactivator). CIITA controls MHC class II expression not only qualitatively, but also quantitatively, and has therefore a direct influence on the CD4 T cell-dependent immune response. CIITA is itself tightly regulated not only on the transcriptional level, but as we show here also on the protein level. CIITA is subjected to a very rapid protein turnover and shows a half-life of about 30,min. Inhibition of degradation by proteasome inhibitors and the identification of ubiquitylated CIITA intermediates indicate that the degradation of CIITA is mediated by the ubiquitin-proteasome system. We identified two regions mediating degradation within the N-terminal domain of CIITA. N-terminal fusions or deletions stabilized CIITA, indicating that the N termini contribute to degradation. Several non-functional CIITA mutants are partially stabilized, but we provide evidence that transcriptional activity of CIITA is not directly linked to degradation. [source] Small Molecule-Controlled Spontaneous Growth of Rose-Like Se Crystals at Room TemperatureEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 7 2008Da-Wei Deng Abstract The spontaneous growth of rose-like Se crystals in aqueous solutions at room temperature is reported. The formation of rose-like Se crystals is based on the oxidation of Na2Se in the presence of thioglycerol solution at pH = 11 in a dark ambient atmosphere. In alkaline solutions, the growth evolution of rose-like Se crystals with aging time was followed by scanning electron microscopy (SEM), and an interesting formation process from initial Se monomers to amorphous Se (a-Se) spheres, and to the final rose-like complex structures of Se crystals was observed. Seven kinds of small molecules with different structures, including 1-thioglycerol (TG), mercaptamine (MA), L -cysteine (L -cys), 3-mercaptopropionic acid (MPA), thioglycolic acid (TGA), glycerol (GLY), and L -serine (L -ser), were used to manipulate the growth of Se crystals. The experimental results show that the structures of the small molecules play a key role in the growth of the Se crystals. The presence of thiols in the structure of the small molecules is favorable for the formation of the aggregates of Se crystals, and other termini, such as ,NH2, ,OH, or ,COO,, will determine whether the aggregates of Se crystals are made up of Se slices or Se prisms. These observations suggest that the ligand molecules have a crucial effect on the nucleation, monomers, and growth of nanocrystals. The selection of ligands can be extended to other important materials for further preparation of nanocrystals with desired shapes. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008) [source] Synthesis and Use of New Substituted 1,3,5-Hexatrienes in Studying Thermally Induced 6, -Electrocyclizations,EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 23 2007Hans Wolf Sünnemann Abstract An acyclic, two heterocyclic, and two bicyclic alkenylstannanes, 3, 4a, 4b, 8 and 11, respectively, were synthesized in yields ranging from 43 to 97,%, and each was subjected to a sequence of Stille and Heck couplings with 2-bromocyclohexenyl triflate (13) and alkyl (tert -butyl and methyl) acrylate to furnish seven new 1,3,5-hexatrienes 19, 20, 21, 22 - tBu, 22 -Me, 23 and 43, respectively, in 58,84,% yields. For the alkenylstannanes 4a,b, 8 and 11, customized combinations of catalysts had to be used. The Stille,Heck sequence involving 13, 3 and tert -butyl acrylate could be performed in a one-pot mode and proceeded in 75,% yield. The hexatrienes were heated in decalin solutions so as to effect 6,-electrocyclization. Temperatures and reaction times were optimized individually. The hexatrienes 29, 31 and 36 gave the bi- and tricyclic cyclohexadienes 28, 30 and 34, incorporating allylic alcohol and allyl ether termini, by 6,-electrocyclization and subsequent [1,5]-hydrogen shift, as single products in good yields (85,93,%). In contrast, the hexatrienes 19, 20, 21 and 39 furnished mixtures of the initial electrocyclization products 26, 32, 37 and 40 as well as the products of a subsequent [1,5]-hydrogen shift 27, 33, 38 and 41, respectively. The tricyclic hexatrienes 22 - tBu, 22 -Me and 23 bearing alkyl (tert -butyl, methyl) acrylate termini also selectively gave the tetracyclic dienes 48 - tBu, 48 -Me and 50 in 71,77,% yields by electrocyclizations and subsequent hydrogen shifts.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source] Preparation of Sugar Amino Acids by Claisen-Johnson Rearrangement: Synthesis and Incorporation into Enkephalin AnaloguesEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 14 2004Ana Montero Abstract We have developed a convenient route for the synthesis of an unsaturated branched sugar bearing a carboxylic acid and an amino group (masked as an azide group) by employing a totally stereoselective Claisen,Johnson rearrangement as the key step. Several Met- and Leu-enkephalin analogues with different substitution patterns at the N - and C -termini were prepared by incorporating this sugar amino acid (SAA) as a substitute for the central Gly,Gly fragment of the parent pentapeptides. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004) [source] Redox-regulated affinity of the third PDZ domain in the phosphotyrosine phosphatase PTP-BL for cysteine-containing target peptidesFEBS JOURNAL, Issue 13 2005Lieke C. J. Van Den Berk PDZ domains are protein,protein interaction modules that are crucial for the assembly of structural and signalling complexes. They specifically bind to short C-terminal peptides and occasionally to internal sequences that structurally resemble such peptide termini. The binding of PDZ domains is dominated by the residues at the P0 and P,2 position within these C-terminal targets, but other residues are also important in determining specificity. In this study, we analysed the binding specificity of the third PDZ domain of protein tyrosine phosphatase BAS-like (PTP-BL) using a C-terminal combinatorial peptide phage library. Binding of PDZ3 to C-termini is preferentially governed by two cysteine residues at the P,1 and P,4 position and a valine residue at the P0 position. Interestingly, we found that this binding is lost upon addition of the reducing agent dithiothrietol, indicating that the interaction is disulfide-bridge-dependent. Site-directed mutagenesis of the single cysteine residue in PDZ3 revealed that this bridge formation does not occur intermolecularly, between peptide and PDZ3 domain, but rather is intramolecular. These data point to a preference of PTP-BL PDZ3 for cyclic C-terminal targets, which may suggest a redox state-sensing role at the cell cortex. [source] The structural comparison of the bacterial PepX and human DPP-IV reveals sites for the design of inhibitors of PepX activityFEBS JOURNAL, Issue 8 2005Pascal Rigolet X-prolyl dipeptidyl aminopeptidases (X-PDAP) are enzymes catalysing the release of dipeptides from the amino termini of polypeptides containing a proline or an alanine at the penultimate position. Involved in various mammalian regulation processes, as well as in chronic human diseases, they have been proposed to play a role in pathogenicity for Streptococci. We compared the structure of X-PDAP from Lactococcus lactis (PepX) with its human counterpart DPP-IV. Despite very different overall folds, the residues most implicated for X-PDAP activity are conserved in the same positions and orientations in both enzymes, thus defining a structural signature for the X-PDAP specificity that crosses the species frontiers of evolution. Starting from this observation, we tested some inhibitors of DPP-IV on PepX activity, for which no specific inhibitor is known. We thus found that PepX was highly sensitive to valine-pyrrolidide with a KI of 9.3 µm, close to that reported in DPP-IV inhibition. We finally used the structure of PepX from L. lactis as a template for computer-based homology modeling of PepX from the pathogenic Streptococcus gordonii. Docking simulations of valine-pyrrolidide into the active site of PepX led to the identification of key residues for a rational drug design against PepX from Streptococci. These results could have applications in human health giving new perspectives to the struggle against pathogens. [source] Extra terminal residues have a profound effect on the folding and solubility of a Plasmodium falciparum sexual stage-specific protein over-expressed in Escherichia coliFEBS JOURNAL, Issue 21 2002Sushil Prasad Sati The presence of extra N- and C- terminal residues can play a major role in the stability, solubility and yield of recombinant proteins. Pfg27 is a 27K soluble protein that is essential for sexual development in Plasmodium falciparum. It was over-expressed using the pMAL-p2 vector as a fusion protein with the maltose binding protein. Six different constructs were made and each of the fusion proteins were expressed and purified. Our results show that the fusion proteins were labile and only partially soluble in five of the constructs resulting in very poor yields. Intriguingly, in the sixth construct, the yield of soluble fusion protein with an extended carboxyl terminus of 17 residues was several fold higher. Various constructs with either N-terminal or smaller C-terminal extensions failed to produce any soluble fusion protein. Furthermore, all five constructs produced Pfg27 that precipitated after protease cleavage from its fusion partner. The sixth construct, which produced soluble protein in high yields, also gave highly stable and soluble Pfg27 after cleavage of the fusion. These results indicate that extra amino acid residues at the termini of over-expressed proteins can have a significant effect on the folding of proteins expressed in E. coli. Our data suggest the potential for development of a novel methodology, which will entail construction of fusion proteins with maltose binding protein as a chaperone on the N-terminus and a C-terminal ,solubilization tag'. This system may allow large-scale production of those proteins that have a tendency to misfold during expression. [source] Casein kinase 2 specifically binds to and phosphorylates the carboxy termini of ENaC subunitsFEBS JOURNAL, Issue 18 2002Haikun Shi A number of findings have suggested the involvement of protein phosphorylation in the regulation of the epithelial Na+ channel (ENaC). A recent study has demonstrated that the C tails of the , and , subunits of ENaC are subject to phosphorylation by at least three protein kinases [Shi, H., Asher, C., Chigaev, A., Yung, Y., Reuveny, E., Seger, R. & Garty, H. (2002) J. Biol. Chem. 277, 13539,13547]. One of them was identified as ERK which phosphorylates ,T613 and ,T623 and affects the channel interaction with Nedd4. The current study identifies a second protein kinase as casein kinase 2 (CK2), or CK-2-like kinase. It phosphorylates ,S631, a well-conserved serine on the , subunit. Such phosphorylation is observed both in vitro using glutathione-S-transferase,ENaC fusion proteins and in vivo in ENaC-expressing Xenopus oocytes. The , subunit is weakly phosphorylated by this protein kinase on another residue (,T599), and the C tail of , is not significantly phosphorylated by this kinase. Thus, CK2 may be involved in the regulation of the epithelial Na+ channel. [source] Substrate specificity of a maize ribosome-inactivating protein differs across diverse taxaFEBS JOURNAL, Issue 7 2000Julie E. Krawetz The superfamily of ribosome-inactivating proteins (RIPs) consists of toxins that catalytically inactivate ribosomes at a universally conserved region of the large ribosomal RNA. RIPs carry out a single N-glycosidation event that alters the binding site of the translational elongational factor eEF1A and causes a cessation of protein synthesis that leads to subsequent cell death. Maize RIP1 is a kernel-specific RIP with the unusual property of being produced as a zymogen, proRIP1. ProRIP1 accumulates during seed development and becomes active during germination when cellular proteases remove acidic residues from a central domain and both termini. These deletions also result in RIP activation in vitro. However, the effectiveness of RIP1 activity against target ribosomes remains species-dependent. To determine the potential efficiency of maize RIP1 as a plant defense protein, we used quantitative RNA gel blots to detect products of RIP activity against intact ribosomal substrates from various species. We determined the enzyme specificity of recombinant maize proRIP1 (rproRIP1), papain-activated rproRIP1 and MOD1 (an active deletion mutant of rproRIP1) against ribosomal substrates with differing levels of RIP sensitivity. The rproRIP1 had no detectable enzymatic activity against ribosomes from any of the species assayed. The papain-activated rproRIP1 was more active than MOD1 against ribosomes from either rabbit or the corn pathogen, Aspergillus flavus, but the difference was much more marked when rabbit ribosomes were used as a substrate. The papain-activated rproRIP1 was much more active against rabbit ribosomes than homologous Zea mays ribosomes and had no detectable effect on Escherichia coli ribosomes. [source] Scanning mutagenesis of regions in the G, protein Gpa1 that are predicted to interact with yeast mating pheromone receptorsFEMS YEAST RESEARCH, Issue 1 2008Douglas P. Gladue Abstract The mechanism by which receptors activate heterotrimeric G proteins was examined by scanning mutagenesis of the Saccharomyces cerevisiae pheromone-responsive G, protein (Gpa1). The juxtaposition of high-resolution structures for rhodopsin and its cognate G protein transducin predicted that at least six regions of G, are in close proximity to the receptor. Mutagenesis was targeted to residues in these domains in Gpa1, which included four loop regions (,2,,3, ,2,,4, ,3,,5, and ,4,,6) as well as the N and C termini. The mutants displayed a range of phenotypes from nonsignaling to constitutive activation of the pheromone pathway. The constitutive activity of some mutants could be explained by decreased production of Gpa1, which permits unregulated signaling by G,,. However, the constitutive activity caused by the F344C and E335C mutations in the ,2,,4 loop and F378C in the ,3,,5 loop was not due to decreased protein levels, and was apparently due to defects in sequestering G,,. The strongest loss of the function mutant, which was not detectably induced by a pheromone, was caused by a K314C substitution in the ,2,,3 loop. Several other mutations caused weak signaling phenotypes. Altogether, these results suggest that residues in different interface regions of G, contribute to activation of signaling. [source] Gene products encoded in the ninR region of phage , participate in Red-mediated recombinationGENES TO CELLS, Issue 4 2002Trudee A. Tarkowski Background:, The ninR region of phage , contains two recombination genes, orf (ninB) and rap (ninG), that were previously shown to have roles when the RecF and RecBCD recombination pathways of E. coli, respectively, operate on phage ,. Results:, When , DNA replication is blocked, recombination is focused at the termini of the virion chromosome. Deletion of the ninR region of , decreases the sharpness of the focusing without diminishing the overall rate of recombination. The phenotype is accounted for in large part by the deletion of rap and of orf. Mutation of the recJ gene of the host partially suppresses the Rap, phenotype. Conclusion: ninR functions Orf and Rap participate in Red recombination, the primary pathway operating when wild-type , grows lytically in rec+ cells. The ability of recJ mutation to suppress the Rap, phenotype indicates that RecJ exonuclease can participate in Red-mediated recombination, at least in the absence of Rap function. A model is presented for Red-mediated RecA-dependent recombination that includes these newly identified participants. [source] Papillary and muscle invasive bladder tumors with distinct genomic stability profiles have different DNA repair fidelity and KU DNA-binding activitiesGENES, CHROMOSOMES AND CANCER, Issue 4 2009Johanne Bentley Low-grade noninvasive papillary bladder tumors are genetically stable whereas muscle invasive bladder tumors display high levels of chromosomal aberrations. As cells deficient for nonhomologous end-joining (NHEJ) pathway components display increased genomic instability, we sought to determine the NHEJ repair characteristics of bladder tumors and correlate this with tumor stage and grade. A panel of 13 human bladder tumors of defined stage and grade were investigated for chromosomal aberrations by comparative genomic hybridization and for NHEJ repair fidelity and function. Repair assays were conducted with extracts made directly from bladder tumor specimens to avoid culture-induced phenotypic alterations and selection bias as only a minority of bladder tumors grow in culture. Four noninvasive bladder tumors (pTaG2), which were genetically stable, repaired a partially incompatible double-strand break (DSB) by NHEJ-dependent annealing of termini and fill-in of overhangs with minimal loss of nucleotides. In contrast, four muscle invasive bladder cancers (pT2-3G3), which displayed gross chromosomal rearrangements, repaired DSBs in an error-prone manner involving extensive resection and microhomology association. Four minimally invasive bladder cancers (pT1G3) had characteristics of both repair types. Error-prone repair in bladder tumors correlated with reduced KU DNA-binding and loss of TP53 function. In conclusion, there were distinct differences in DSB repair between noninvasive papillary tumors and higher stage/grade invasive cancers. End-joining fidelity correlated with stage and was increasingly error-prone as tumors became more invasive and KU binding activity reduced; these changes may underlie the different genomic profiles of these tumors. © 2008 Wiley-Liss, Inc. [source] Comprehensive karyotyping of the HT-29 colon adenocarcinoma cell lineGENES, CHROMOSOMES AND CANCER, Issue 1 2002Kanji Kawai The tumor cell line HT-29 was derived from a primary adenocarcinoma of the rectosigmoid colon. HT-29 is hypertriploid (3n+) and has accumulated numerous chromosomal structural aberrations. To identify material involved in chromosome rearrangements, we performed a comprehensive cytogenetic analysis using G-banding, spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH). The combination of molecular cytogenetic techniques enabled us to define the first comprehensive karyotype for HT-29. Seventeen marker chromosomes were found in 75,100% of metaphase cells, generally in a single copy per cell. We confirmed the composition of eight previously described markers, refined the classification of seven others, and identified two novel marker chromosomes. Notable aberrations included a reciprocal translocation between chromosomes 6 and 14 and an unusual, large derivative chromosome 8 composed entirely of 8q material. The telomere status, evaluated by FISH, revealed telomeric signals at the termini of all chromosomes. No interstitial telomeric sequences were observed in any cell. Although numerous chromosomal aberrations are present in HT-29, the cell line appears to have retained a high level of genomic stability during passage in culture since undergoing transformation. The excellent resolving power of SKY, coupled with additional information obtained from molecular cytogenetic analyses, will improve our ability to identify genetic lesions characteristic of cancer. © 2002 Wiley-Liss, Inc. [source] Tyrosine phosphorylation of the GluR2 subunit is required for long-term depression of synaptic efficacy in young animals in vivoHIPPOCAMPUS, Issue 8 2007Christopher J. Fox Abstract The study of the intracellular mechanics that underlay changes in synaptic efficacy is a rapidly evolving field of research. It is currently believed that NMDA receptors play a significant role in the induction of synaptic plasticity, whereas AMPA receptors play a significant role in its expression. For AMPA receptors, it has been shown that tyrosine phosphorylation of the GluR2 carboxyl termini is required for the expression of long-term depression of synaptic efficacy (LTD) in vitro (Ahmadian et al. (2004) EMBO J 23:1040,1050). In the present study, we sought to determine whether similar mechanisms are involved in vivo, where different stimulation parameters are required for the induction of LTD. We initially used a paired-burst (PB) paradigm that reliably induces LTD in vivo. In these animals we were able to prevent the induction and expression of PB-LTD by administering a peptide (GluR-3Y) that acted as a competitive inhibitor of tyrosine phosphorylation. In a separate set of animals, we exposed animals to brief periods of stress (S) before using low-frequency stimuli to induce LTD (S-LTD). Again, GluR2,3Y blocked both the induction and expression of S-LTD. In contrast, an inert version of the peptide, with alanine replacing the three tyrosine residues, did not inhibit LTD induction. In addition, we demonstrated that GluR2,3Y did not affect the induction of long-term potentiation in vivo. These findings support the hypothesis that tyrosine phosphorylation and AMPA receptor endocytosis are necessary steps for the induction and maintenance of two forms of LTD in the CA1 region. © 2007 Wiley-Liss, Inc. [source] Lafora disease in the Indian population: EPM2A and NHLRC1 gene mutations and their impact on subcellular localization of laforin and malin,HUMAN MUTATION, Issue 6 2008Shweta Singh Abstract Lafora disease (LD) is a fatal form of teenage-onset autosomal recessive progressive myoclonus epilepsy. LD is more common among geographic isolates and in populations with a higher rate of consanguinity. Mutations in two genes, EPM2A encoding laforin phosphatase, and NHLRC1 encoding malin ubiquitin ligase, have been shown to cause the LD. We describe here a systematic analysis of the EPM2A and the NHLRC1 gene sequences in 20,LD families from the Indian population. We identified 12 distinct mutations in 15,LD families. The identified novel mutations include 4 missense mutations (K140N, L310W, N148Y, and E210,K) and a deletion of exon 3 for EPM2A, and 4 missense mutations (S22R, L279P, L279P, and L126P) and a single base-pair insertional mutation (612insT) for NHLRC1. The EPM2A gene is known to encode two laforin isoforms having distinct carboxyl termini; a major isoform localized in the cytoplasm, and a minor isoform that targeted the nucleus. We show here that the effect of the EPM2A gene mutation L310W was limited to the cytoplasmic isoform of laforin, and altered its subcellular localization. We have also analyzed the impact of NHLRC1 mutations on the subcellular localization of malin. Of the 6 distinct mutants tested, three targeted the nucleus, one formed perinuclear aggregates, and two did not show any significant difference in the subcellular localization as compared to the wild-type malin. Our results suggest that the altered subcellular localization of mutant proteins of the EPM2A and NHLRC1 genes could be one of the molecular bases of the LD phenotype. © 2008 Wiley-Liss, Inc. [source] Towards Control of Dendrimer Properties by Reversible Exchange of Termini: Synthesis and Characterization of Diverse Porphyrin DendrimersISRAEL JOURNAL OF CHEMISTRY, Issue 1 2009Meital Shema-Mizrachi Porphyrin dendrimers with boronic ester, aldehyde, and pyridil termini were synthesized and fully characterized. These dendrimers have the potential to change their physical and chemical properties by reversible alteration of the reactive terminal groups. [source] Two C-Terminal Variants of NBC4, a New Member of the Sodium Bicarbonate Cotransporter Family: Cloning, Characterization, and LocalizationIUBMB LIFE, Issue 1 2000Alexander Pushkin Abstract We report the cloning, characterization, and chromosomal assignment of a new member of the sodium bicarbonate cotransporter (NBC) family, NBC4. The NBC4 gene was mapped to chromosome 2p13 and is a new candidate gene for Alstrom syndrome. Two variants of the transporter have been isolated from human testis and heart, which differ in their C termini. NBC4a encodes a 1137-residue polypeptide and is widely expressed in various tissues, including liver, testis, and spleen. NBC4b is identical to NBC4a except that it has a 16-nucleotide insert, creating a C-terminal frame shift. NBC4b encodes a 1074-residue polypeptide and is highly expressed in heart. Amino acids 1-1046 are common to both NBC4 variants. NBC4a has two protein-interacting domains that are lacking in NBC4b: a proline-rich sequence, PPPSVIKIP (amino acids 1102-1110), and a consensus PDZ-interacting domain, SYSL (1134-1137). NBC4b lacks the stretch of charged residues present in the C terminus of NBC4a and other members of the NBC family.Unlike other members of the NBC family, both NBC4a and NBC4b have a unique glycine-rich region (amino acids 440- 469). In comparison with other members of the bicarbonate transport superfamily, NBC4a and NBC4b are most similar structurally to the electrogenic sodium bicarbonate cotransporters (NBC1). [source] | ||||||||||||||||||||||||||||||||||||||||||||||||||||