bluuebunny/crossref_metadata_2025 huggingface dataset:
Thyroid hormone modulates the extracellular matrix organization and expression in cerebellar astrocyte: Effects on astrocyte adhesion
Authors: Gonçalves Trentin Andréa , De Aguiar Cláudia Beatriz Nedel Mendes , Castilho Garcez Ricardo , Alvarez‐Silva Marcio
Published: April 2003

The effects of thyroid hormone (T3) on extracellular matrix (ECM) expression and organization in cerebellar astrocytes were studied. Control astrocytes exhibit laminin immunostaining distributed in a punctate configuration and fibronectin concentrated in focal points at the cell surface. These cells attach to the substratum by membrane points, as shown by scanning microscopy, possibly by focal points stained to fibronectin. In contrast, after T3 treatment, laminin assumes a fibrillary pattern and fibronectin becomes organized in filaments homogeneously distributed on the cell surface; the cells acquire a very flat and spread morphology. T3 treatment also modulates astrocyte adhesion. In addition, increased expression of both laminin and fibronectin was detected by Western blot. These alterations in fibronectin and/or laminin production and organization may be involved in the flat and spread morphology and in altered adhesion. We observed that fibroblast growth factor‐2 (FGF2) added to cultures had similar effects to those described to T3. Neutralizing antibodies against FGF2 reversed T3 effects on fibronectin and laminin distribution. We also observed that cerebellar neurons co‐cultured on T3‐treated astrocytes had an increase in the number of cells and presented longer neurites. Thus, we propose a novel mechanism of the effect of thyroid hormone on cerebellar development mediated by astrocytes: T3 may induce astrocyte secretion of growth factors, mainly FGF2, that autocrinally stimulate astrocyte proliferation, reorganization in ECM proteins, and alterations in cell spreading and adhesion. These effects may indirectly influence neuronal development. GLIA 42:359–369, 2003. © 2003 Wiley‐Liss, Inc.
Synthesis of a Tridentate Ligand for Use in TiIV‐Catalyzed Acetate Aldol Addition Reactions
Authors: Singer Robert A. , Brock Jane R. , Carreira Erick M.
Published: April 2003

A facile, practical synthesis and resolution of (±)‐2′‐amino‐[1,1′‐binaphthalen]‐2‐ol (1) is described, as well as the preparation of the tridentate Schiff base ligand 2 derived from condensation of 1 with 3‐bromo‐5‐(tert‐butyl)salicylaldehyde, which has been used in catalytic enantioselective acetate aldol addition reactions.
Myth and Reality in the Attitude toward Valence‐Bond (VB) Theory: Are Its ‘Failures’ Real?
Authors: Shaik Sason , Hiberty Philippe C.
Published: April 2003

According to common wisdom propagated in textbooks and papers, valence‐bond (VB) theory fails and makes predictions in contradiction with experiment. Four iconic ‘failures’ are: a) the wrong prediction of the ground state of the O2 molecule, b) the failure to predict the properties of cyclobutadiene (CBD) viz. those of benzene, c) the failure to predict the aromaticity/anti‐aromaticity of molecular ions like C5H$\rm{_{5}^{+}}$ and C5H$\rm{_{5}^{-}}$, C3H$\rm{_{3}^{+}}$ and C3H$\rm{_{3}^{-}}$, C7H$\rm{_{7}^{+}}$ and C7H$\rm{_{7}^{-}}$, etc; and d) the failure to predict that, e.g., CH4 has two different ionization potentials. This paper analyzes the origins of these ‘failures’ and shows that two of them (stated in a and d) are myths of unclear origins, while the other two originate in misuse of an oversimplified version of VB theory, i.e., simple resonance theory that merely enumerate resonance structures. It is demonstrated that, in each case, a properly used VB theory at a simple and portable level leads to correct predictions, as successful as those made by use of molecular‐orbital (MO) theory. This notion of VB ‘failure’, which is traced back to the VB‐MO rivalry, in the early days of quantum chemistry, should now be considered obsolete, unwarranted, and counterproductive. A modern chemist should know that there are two ways of describing electronic structure, which are not two contrasting theories, but rather two representations or two guises of the same reality. Their capabilities and insights into chemical problems are complementary, and the exclusion of any one of them undermines the intellectual heritage of chemistry.
Cocrystallization with Acetylene. The 1 : 1 Complex with Benzene: Crystal Growth, X‐Ray Diffraction and Molecular Simulations
Authors: Boese Roland , Clark Timothy , Gavezzotti Angelo
Published: April 2003

The crystal structure of an unusual 1 : 1 molecular complex between benzene and acetylene, two very small and apolar molecules, has been determined by X‐ray‐analysis of crystals grown by first mixing the two liquids under conditions of low temperature and high pressure in a capillary, followed by repeated zone melting to form crystals directly on the goniometer head of a diffractometer. Each acetylene molecule is clamped between two parallel benzene rings, with its molecular axis apparently perpendicular to the benzene planes. Closer inspection of thermal‐motion data from the diffraction experiment suggests that the acetylene molecule undergoes a wobbling, or precession, motion between the two rings so that it is perpendicular to them only in a time‐averaged sense. The results of quantum‐chemical calculations on isolated molecular dimers and trimers support this conclusion. In addition, the calculation of separate coulombic, dispersion, polarization and repulsion contributions to intermolecular bonding reveals that the CH⋅⋅⋅π‐bond interaction between acetylene and benzene in a T‐shaped dimer consists of a mixture of coulombic and polarization interactions. In the benzeneacetylene cocrystal, its magnitude is quantitatively comparable with that of other dispersive interactions. 5.4 ns Molecular‐dynamics simulations of the liquid mixture reveal that the two components are persistently miscible, a possible key to the formation of the cocrystal. No structure is, however, observed in the solution during the relatively short simulation time.
Structural Correlations for Nucleophilic Addition to the CO Group: The Solvation Angle
Authors: Davies John E. , Kirby Anthony J. , Komarov Igor V.
Published: April 2003

We report some new observations in BürgiDunitz territory – structurestructure correlations for the addition of N nucleophiles to the CO group. The amino aldehyde 1 exists predominantly in polar solvents, and exclusively in the crystal, as the carbonyl‐addition structure 2, but appears to demand hydrogen‐bonding solvation as part of the process. In terms of CO bond lengthening, the new system lies somewhere nearly half‐way between an amino aldehyde and a stable quaternary ammonium aminal: we present the structure 2⋅Me of the N+COMe derivative of 2 as an example. We have investigated the effects on the cyclisation equilibrium in this system of hydration, which is important, and of the degree of methylation of the azaadamantane skeleton, which – unexpectedly – is not. In terms of the reverse process, i.e., breaking the CN+ bond: in each case, the bond is lengthened by (n(O)σ*(CN)) electron‐pair donation from the O‐atom (the generalised anomeric effect, as in 2⋅Me↔3, below) [4]. This effect is strongest when the O‐atom is negatively charged, reduced by hydrogen‐bonding, and minimised by alkylation.
Crystal Structures and Magnetic Properties of Nitronyl Nitroxide Radicals
Authors: Zakrassov Alexander , Shteiman Vitaly , Sheynin Yana , Botoshansky Mark , Kapon Moshe , Kaftory Menahem , Del Sesto Rico E. , Miller Joel S.
Published: April 2003

The crystal structures and magnetic properties of the nitronyl nitroxide radicals 4,5‐dihydro‐4,4,5,5‐tetramethyl‐3‐oxido(1H‐imidazol‐1‐yloxyl) (1), 4,5‐dihydro‐2,4,4,5,5‐pentamethyl‐3‐oxido(1H‐imidazol‐1‐yloxyl) (2), 2‐(4‐chlorophenyl)‐4,5‐dihydro‐4,4,5,5‐tetramethyl‐3‐oxido(1H‐imidazol‐1‐yloxyl) (3), and 4,5‐dihydro‐2‐(2‐hydroxy‐5‐nitrophenyl)‐4,4,5,5‐tetramethyl‐3‐oxido(1H‐imidazol‐1‐yloxyl) (4) are reported. Compound 1 has two polymorphic forms: the α phase is monoclinic (P21/n space group), with a single molecule in the asymmetric unit, and the β phase is monoclinic (P21/c space group), with four molecules in the asymmetric unit. In the two polymorphs, the molecules are arranged in dimers formed by hydrogen bonds of the type CH⋅⋅⋅ON. The crystal structure of 3 contains layers of antiparallel ribbons of molecules. Compound 4 crystallizes with solvent molecules, and an intramolecular hydrogen bond is formed between the 2‐OH group of the phenyl ring and the nitroxide O‐atom. Compound 4 also loses the two O‐atoms of the nitroxide moiety upon heating to 90°. Magnetic measurements showed that both α and β polymorphs of 1 exhibit antiferromagnetic coupling. The best fit to the experimental data was obtained using BleanyBower's singlet‐triplet model (H=−2JSaSb): J=−11.2 K for the α phase and J=−15.0 K for the β phase. Compounds 3 and 4 show no evidence for spin coupling.
Distorted Fivefold‐Diamond Structure of 4,4‐Bis(2‐carboxyethyl)pimelic Acid (‘Methanetetrapropionic Acid’)
Authors: Ermer Otto , Kusch Andreas , Röbke Christof
Published: April 2003

The molecules of 4,4‐bis(2‐carboxyethyl)pimelic acid (=4,4‐bis(2‐carboxyethyl)heptanedioic acid; 1) assemble to build up hollow, five‐fold interpenetrating diamond‐like networks in tetragonal crystals via standard pairwise H‐bonds between the carboxy groups. In keeping with the flat, extended molecular conformation of 1 of approximate D2d symmetry, the supramolecular diamondoid networks are tetragonally compressed. Their mode of interpenetration is unusual and corresponds to that of adamantane‐1,3,5,7‐tetracarboxylic acid, which has an undistorted five‐fold diamond structure. Both the degree and the mode of interpenetration of 1 differ from those of the lower homologue 3,3‐bis(carboxymethyl)glutaric acid (2), which adopts a more‐common tetragonally elongated triple‐diamond architecture in the solid state.
The History of Ozone. Part III
Authors: Rubin Mordecai B.
Published: April 2003

The introduction of ozone into organic chemistry is due to the work of C. D. Harries, first in Berlin but mainly in Kiel, during the period 1903–1916. He established experimental procedures for ozonolysis, demonstrated the generality of the reaction of unsaturated compounds with ozone, and showed that ozone could be used for the synthesis of a variety of sensitive compounds. Most important for the future development of organic chemistry, he established that ozonolysis could be an important tool for determining the structures of organic compounds. He also initiated investigations into the mechanism of ozone reactions with alkenes, which culminated about forty years later in the Criegee mechanism for ozonolysis.
Polyacetylenes and Cumulenes, Potential Elements for Molecular Machines and Precursors of Carbon Clusters: A Theoretical Study
Authors: Zahradník Rudolf , Šroubková Libuše
Published: April 2003

A recently described very simple procedure for estimating Hartree–Fock (H. F.) energy and accurate nonrelativistic energy has been used with simple hydrocarbons possessing CC or CC bonds and for the acetylene dimer. Experimental characteristics (heats of formation, ionization potentials, electron affinities), structural features, and reactivity have been discussed in terms of quantum‐chemistry characteristics at the H. F. level and also at a level including a part of electron correlation. Deviations from linearity with derivatives of long polyacetylenes and cumulenes are ascribed to the lowest‐energy deformation vibrations, which decrease rapidly when passing from short to long acetylenic and cumulenic chains. The role of derivatives and heteroanalogues of hydrocarbons under study in atmospheric and interstellar chemistry is briefly mentioned. Derivatives with enhanced stability represent potentially promising construction materials for molecular devices and also resources for the preparation of defined clusters of C‐atoms.
Parental assessment of early childhood development: biological and social covariates
Authors: Pevalin David J. , Wade Terrance J. , Brannigan Augustine
Published: April 2003

This study uses data from the National Longitudinal Survey of Children and Youth (N=8605) to investigate the effects of unfavourable birth outcomes and dimensions of the family environment on the development of Canadian children aged 0–47 months. Among children under 24 months of age, it was found that sex of the child, birth order and low birthweight were significant covariates while the familial environment tended to have little influence. However, among children aged 24–47 months, effects of parenting style, social support, and positive social‐structural conditions of the family supplemented the effects of sex of the child and low birthweight. The results suggest that the cumulative effects of a positive family environment begin to occlude the neonatal disadvantages in the first 47 months of life. Copyright © 2003 John Wiley & Sons, Ltd.
Mass Spectrometric Characterization of a New 2‐Hydroxypropyl‐β‐cyclodextrin Derivative Bearing Methacrylic Moieties and Its Copolymerization with 1‐Vinyl‐2‐pyrrolidone
Authors: Janus Ludovic , Carbonnier Benjamin , Morcellet Michel , Ricart Guy , Crini Grégorio , Deratani André
Published: April 2003

The characterization of a new cyclodextrin (CD) derivative, developed by Wacker Chemie GmbH under the name βW7‐MAHP and consisting of methacrylic moieties attached to hydroxypropyl‐β‐CD units, was investigated in terms of molecular structure and reactivity. The structural study was performed using a matrix‐assisted laser‐desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometry analysis, which revealed that βW7‐MAHP is a mixture of at least 60 identified compounds. The substituent nature was determined from the two successive chemical modifications performed on β‐CD (hydroxypropylation with propylene oxide followed by the reaction with glycidyl methacrylate). Furthermore, each reaction resulted in a mixture of compounds having different numbers of substituents because of the polyfunctionality of the β‐CD molecule (14 secondary and 7 primary OH groups). Only 50 wt.‐% of the mixture consisted of polymerizable components among which a small proportion contained two or more methacrylic residues. The polymerizability of βW7‐MAHP was studied, and the reactivity ratios in the copolymerization with 1‐vinyl‐2‐pyrrolidinone were estimated. Soluble copolymers were obtained for monomer compositions having a βW7‐MAHP fraction less than 60 mol‐% whereas for higher amounts cross‐linked gels were produced.

MALDI‐TOF mass spectrum of βW7‐MAHP.

magnified image

MALDI‐TOF mass spectrum of βW7‐MAHP.
The configurational characteristics of conventional poly(methyl methacrylate) in acetonitrile
Authors: Quadrat Otakar , Bohdanecký Miloslav , Mrkvičková Libuše
Published: April 2003

The θ‐temperature and viscosity constant K0 of radically initiated poly(methyl methacrylate) in acetonitrile were determined from the intrinsic viscosity data (θ = 28°C, K0 = 4,3 · 102 g−3/2 · mol1/2.cm3). The unperturbed coil dimensions are lower in acetonitrile than in other θ‐solvents at comparable temperature and indicate a more compact conformation of poly(methyl methacrylate) molecules. The discrepancies in the reported data of the θ and K0 values are elucidated.
Polymerization of Methyl Methacrylate Initiated by a Divalent Samarium Phenoxide Complex with an Alkyl Aluminium Compound
Authors: Nodono Mitsufumi , Tokimitsu Toru , Makino Takayuki
Published: April 2003

A divalent samarium complex that bears two bulky phenoxide ligands promotes the living polymerization of methyl methacrylate in the absence of cocatalyst. The initiation involves a tail‐to‐tail coupling reaction of the samarium‐enolate radical followed by a one‐electron reduction of the monomer with the divalent samarium initiator. The polymerization takes place stereospecifically and the samarium initiator affords highly syndiotactic poly(methyl methacrylate). The stereoregularity of the polymer is variable with the ratio of the samarium initiator to aluminium compound (2,6‐di‐tert‐butyl‐4‐methylphenoxy)2AlMe. The polymer obtained when the [Al]/[Sm] ratio is 4.0 has a unique tacticity, [mm]/[mr]/[rr] = 42:11:47, and the polymer has a melting point at 161 °C, which suggests the formation of a stereocomplex between syndiotactic and isotactic poly(methyl methacrylate).

Part of the possible initiation mechanism and equimolar reaction of BHT‐SmII with MMA.

magnified image

Part of the possible initiation mechanism and equimolar reaction of BHT‐SmII with MMA.
Homo‐ and Copolymerization of Norbornene and Styrene with Pd‐ and Ni‐Based Novel Bridged Dinuclear Diimine Complexes and MAO
Authors: Mi Xia , Ma Zhi , Wang Leyong , Ke Yucai , Hu Youliang
Published: April 2003

Novel bridged dinuclear diimine nickel and palladium complexes catalyzed the vinyl addition polymerization of norbornene at a single active site with high activity. The latter exhibited higher activity than the former. Smaller substituents around the imido group in the ligand were more favorable for increasing the activity than bulkier ones. The polynorbornenes were all noncrystalline but had short‐range order and their glass transition temperature, Tg, ranged from 270 to 400 °C. Furthermore, the random copolymerization of norbornene and styrene was catalyzed by the bridged dinuclear diimine nickel complex with MAO (methylaluminoxane). Gel permeation chromatography (GPC), FTIR and 1H NMR spectroscopies, wide‐angle X‐ray diffraction (WAXD), and differential scanning calorimetry (DSC) indicated that the copolymers are “true” copolymers. The reactivity ratios were rstyrene = 0.281, rnorbornene = 18.7. The packing density, thermostability and the glass transition temperatures of copolymers are increased relative to polystyrene. The solubility and processability of the copolymers are improved relative to polynorbornene.

The structures of bridged dinuclear diimine nickel and palladium complexes.

magnified image

The structures of bridged dinuclear diimine nickel and palladium complexes.
Transition metal catalysts. VIII. The role of oxygen in ethylene polymerizations with the AlBr3‐VXn‐Sn(C6H5)4 catalyst
Authors: Phillips George W. , Carrick Wayne L.
Published: April 2003

Ethylene polymerizations with the aged AlBr3–VXn–Sn(C6H5)4 catalyst required the presence of about 0.02% oxygen in the ethylene feed. When the catalyst components were combined in the presence of ethylene, polymerization ceased in less than 1 min. with the production of about one polymer molecule per vanadium atom. The reaction was repeatedly reactivated by the addition of milligram amounts of oxygen, ultimately resulting in a higher yield of polyethylene with a molecular weight of 230,000. In the concentration range from 0.003 to 6% oxygen in the ethylene, there was a sharp maximum in the amount of polymer produced, before polymerization ceased, at about 0.05–0.1% oxygen. However, the molecular weight of the polyethylene decreased mono‐tonically with increasing oxygen concentration from 230,000 at 0.003% to 12,500 at 6.1% oxygen. It was demonstrated that oxygen enters into an oxidation‐reduction cycle with the vanadium species and that this reaction induces molecular termination and reactivates the vanadium center. In the absence of oxygen, the polyethylene was found to contain one phenyl and one vinyl endgroup per polymer molecule. These data are interpreted in light of a previously proposed propagation mechanism. Polymer end group analyses suggest that termination occurs by transfer of the polymer from the active vanadium center to an inactive aluminum center when oxygen is absent and by oxidation of the vanadium active site to a catalytically inactive compound when oxygen is added.
When Gold Is Not Noble: Catalysis by Nanoparticles
Authors: Haruta Masatake
Published: April 2003

Bulk gold is chemically inert and is generally regarded as a poor catalyst. However, when gold is in very small particles with diameters
below 10 nm and is deposited on metal oxides or activated carbon, it becomes surprisingly active, especially at low temperatures, for
many reactions such as CO oxidation and propylene epoxidation. The catalytic performance of Au is defined by three major factors: contact structure,
support selection, and particle size. The role of the perimeter interfaces of Au particles as the sites for reactions is discussed as well as the
change in chemical reactivity of Au clusters composed of fewer than 300 atoms. © 2003 The Japan Chemical Journal Forum and Wiley Periodicals,
Inc., Chem Rec 3: 75–87; 2003: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.10053
Determination of Molecular Stereochemistry Using Vibrational Circular Dichroism Spectroscopy: Absolute Configuration and Solution Conformation of 5‐Formyl‐cis, cis‐1,3,5‐trimethyl‐3‐hydroxymethylcyclohexane‐1‐carboxylic Acid Lactone
Authors: Izumi Hiroshi , Futamura Shigeru , Nafie Laurence A. , Dukor Rina K.
Published: April 2003

The determination of the absolute configuration of chiral molecules is an important aspect of molecular stereochemistry. Vibrational
circular dichroism (VCD) is the extension of electronic CD into the infrared region where fundamental vibrational transitions
occur. VCD has a number of advantages over all previous methods of absolute configuration assignment. The absolute configuration and
predominant solution‐state conformation in CDCl3 of the chiral lactone,
5‐formyl‐cis,cis‐1,3,5‐trimethyl‐3‐hydroxymethylcyclohexane‐1‐carboxylic
acid lactone, 1, obtained by the comparison of measured and calculated VCD spectra, are reported. It is found that
(−)‐1 corresponds to the absolute configuration (1S,3S,5R)‐1.
© 2003 The Japan Chemical Journal Forum and Wiley Periodicals, Inc., Chem Rec 3: 112–119; 2003: Published online in Wiley
InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.10055
Down‐regulation of phospholipase C‐γ1 during the differentiation of U937 cells
Authors: Lee Young Han , Lee He Jin , Lee Seung-Jae , Min Do Sik , Baek Suk Hwan , Kim Yong Sik , Ryu Sung Ho , Suh Pann-Ghill
Published: April 2003

Phospholipase C (PLC)‐γ1 and ‐γ2 play a pivotal role in signal transduction for cell proliferation and differentiation. The enzyme activity and protein level of PLC‐γ1 were markedly decreased in the human histiocytic leukemia U937 cell line during the differentiation process which is induced by phorbol 12‐myristate 13‐acetate (PMA) but those of PLC‐γ2 were not altered. Northern blot analysis showed that the levels of PLC‐γ1 and ‐γ2 transcripts were not changed. These results suggest that the expression of PLC‐γ1 during the PMA‐induced differentiation may be down‐regulated by post‐transcriptional processing.
Interaction between hsp70 and hsp40, eukaryotic homologues of DnaK and DnaJ, in human cells expressing mutant‐type p53
Authors: Sugito Kazuhiro , Yamane Mitsuo , Hattori Hirotomo , Hayashi Yasushi , Tohnai Iwai , Ueda Minoru , Tsuchida Nobuo , Ohtsuka Kenzo
Published: April 2003

We have recently identified a novel 40‐kDa heatshock protein hsp40 as a mammalian homologue of bacterial DnaJ protein. Here we demonstrate the physical interaction between hsp70 (DnaK homologue) and hsp40 in human cells as determined by immunoprecipitation methods. Co‐immunoprecipitation of hsp70 with hsp40 was dependent on the presence of ATP or unfolded protein (reduced carboxymethylated α‐lactalbumin). A mutant type of tumor suppressor gene product, mtp53, was co‐immunoprecipitated not only with hsp70 but also with hsp40. These results suggest the existence of a hsp70(DnaK)/ hsp40(DnaJ) chaperone system in mammalian cells.
Enhancing peptide antigenicity by helix stabilization
Authors: Gurunath Ramanathan , Beena T.K. , Adiga P.R. , Balaram P.
Published: April 2003

The engineering of antigenic determinants on super secondary structures using de novo design approaches often involves synthesis of long peptide chains (35–80 residues long). This communication illustrates that the stabilization of secondary structure by rational design can also greatly enhance immunogenicity and antigenicity, but in much shorter peptide sequences (21 residues long). A peptide epitope the sequence of which has been derived from the C‐terminus of the chicken riboflavin carrier protein (cRCP), H2N‐Tyr‐His‐Ala‐Cys‐Gln‐Lys‐Lys‐Leu‐Leu‐ Lys‐Phe‐Glu‐Ala‐Leu‐Gln‐Gln‐Glu‐Glu‐Gly‐Gly‐Glu‐Glu‐OH, has been chosen for analysis. Helical conformations were induced in the peptide in aqueous trifluoroethanol. Analogs were designed to stabilize this conformation in water by either the introduction of appropriately spaced ion pairs or the strongly helix nucleating residue α‐aminoisobutyric acid (Aib), substituted for Ala/Gly, thus affording a comparison of the helix stabilization strategies. Circular dichroism (CD) results demonstrate that all the designed analogs are appreciably more helical than the parent peptide in 50% aqueous trifluoroethanol. Peptide antisera were raised for all analogs in rabbits. The affinities of these antisera for the native protein antigen, determined using a chaotrope disrupted binding assay, correlated very well with the helix content determined by CD.
Temporary inhibition of papain by hairpin loop mutants of chicken cystatin Distorted binding of the loops results in cleavage of the Gly9‐Ala10 bond
Authors: Machleidt Werner , Nägler Dorit K. , Assfalg-Machleidt Irmgard , Stubbs Milton T. , Fritz Hans , Auerswald Ennes A.
Published: April 2003

Temporary inhibition of the cysteine proteinases papain and cathepsin L was observed with several hairpin loop mutants of recombinant chicken cystatin at enzyme concentrations above nanomolar. Kinetic modelling of inhibition data, gel electrophoresis and amino acid sequencing revealed that reappearance of papain activity is due to selective cleavage of the Gly9‐Ala10 bond in the N‐terminal binding area of the chicken cystatin variants, resulting in truncated inhibitors of lower affinity. Cleavage of the same bond by contaminating papaya proteinase IV was ruled out by previous purification of papain and suitable control experiments. According to the proposed kinetic model, cleavage occurs within the enzyme‐inhibitor complex with first order rate constants k
temp of 2.3 × 10−3 up to 5 × 10−1 s−1. A similar ratio was found for all mutants (0.7 × 106–2.1 × 106 s−1·M−1); it is almost identical with the ratio of the peptide substrate Z‐Phe‐Arg‐NHMec. These results suggest that distorted contacts of one of the hairpin loops affect binding of the N‐terminal contact area in a way that covalent interaction of the Gly9‐Ala10 bond with the active‐site Cys residue of papain can occur and the bond is cleaved in a substrate‐like manner.
Hairpin loop mutations of chicken cystatin have different effects on the inhibition of cathepsin B, cathepsin L and papain
Authors: Auerswald Ennes A. , Nägler Dorit K. , Assfalg-Machleidt Irmgard , Stubbs Milton T. , Machleidt Werner , Fritz Hans
Published: April 2003

Five recombinant hairpin loop variants of chicken cystatin (ΔV55, ΔV55‐S56, ΔP103‐L105, ΔI102‐Q107, loop2‐KD2) were constructed by cassette mutagenesis, expressed in E. coli, purified to homogeneity, characterized by protein‐chemical means and by their inhibitory properties. The variant forms, modified in two of the three postulated cysteine proteinase binding regions, were inhibitorily active. However, the equilibrium dissociation constants of the complexes between papain as well as human cathepsin B or L and the cystatin variants show a weaker affinity for all three enzymes compared with recombinant chicken cystatin. These results prove the contribution of both hairpin loops to complex formation with the three enzymes. Furthermore, the kinetic constants indicate discrete differences in the molecular mechanism of interaction between chicken cystatin and papain, cathepsin B, and cathepsin L. Inhibition of cathepsin L was much less affected than inhibition of papain or cathepsin B by the modifications achieved in the five variants. Remarkably, at high enzyme concentration (above 0.5 nM) inhibition of papain by these variants was ‘temporary’, that means, active papain was released from the enzyme‐inhibitor complex within minutes to hours (compare [1]).
Synaptotagmin V: a novel synaptotagmin isoform expressed in rat brain
Authors: Craxton Molly , Goedert Michel
Published: April 2003

Regulated Ca2+‐dependent release of transmitters from synaptic vesicles is an important characteristic of chemical neurotransmission. Synaptotagmins are abundant synaptic vesicle transmembrane proteins that probably function as Ca2+ sensors. Molecular cloning has identified four different synaptotagmin isoforms in mammals. We report here the cloning and sequencing of a novel isoform of 386 amino acids. Synaptotagmin V is 54% identical in sequence to synaptotagmin I and possesses all the domains that characterise this multigene family. It is expressed at high levels in rat brain, but not in spinal cord or a number of peripheral non‐neuronal tissues.
Cloning and sequencing of the bacterioferritin gene of Brucella melitensis 16M strain
Authors: Denoel Philippe A. , Zygmunt Michel S. , Weynants Vincent , Tibor Anne , Lichtfouse Bernadette , Briffeuil Pascal , Limet Joseph N. , Letesson Jean-Jacques
Published: April 2003

The 40 N‐terminal amino acids of the 20 kDa antigen A2 from Brucella melitensis were sequenced and showed important similarities with 4 bacterioferritins. A monoclonal antibody raised against this antigen cross‐reacted with Escherichia coli bacterioferritin. Hybridization of two sets of degenerate primers with B. melitensis HindIII‐digested genomic DNA identified a 3.8 kb fragment. This fragment was shown to contain a bacterioferritin gene (bfr) encoding a 161‐amino acid protein. The sequence of the Brucella bacterioferritin is 69% similar to that of E. coli, and many of the ferroxidase centre and haem‐ligation residues are conserved.
EPR spectroscopy of 5‐DOXYL‐stearic acid bound to the mitochondrial uncoupling protein reveals its competitive displacement by alkylsulfonates in the channel and allosteric displacement by ATP
Authors: Ježek Petr , Bauer Michael , Trommer Wolfgang E.
Published: April 2003

Competition of fatty acids (FA) and alkylsulfonates with 5‐DOXYL‐stearic acid (5‐SASL) binding to isolated mitochondrial uncoupling protein (UcP) is demonstrated using EPR spectroscopy. A distinct peak of the bound 5‐SASL (h+1I) decreased with increasing concentration of competitors. Since alkylsulfonates are UcP substrates, it suggests that the FA binding site is located in the anion channel. Moreover, with increasing ATP the h+1I peak decreased and was smoothed with the ‘micellar’ peak into a single wider peak. A pH of 8.5 reversed this effect. It could reflect an allosteric release of 5‐SASL from the ATP binding site which mimics the ATP gating mechanism.
Studies on the promoter of the Arabidopsis thaliana cdc2a gene
Authors: Chung Susanna K. , Parish Roger W.
Published: April 2003

The 5′ flanking (promoter) region of the Arabidopsis thaliana cdc2a gene was cloned and sequenced. A number of putative regulatory motifs were identified including one Myc and three Myb protein binding sequences plus one abscisic acid and two auxin responsive elements. One of the three Myb protein binding sequences is positioned within an auxRE. Promoter‐GUS fusions were introduced into plants to study the role of two promoter regions in regulating gene expression. Absence of one Myb binding sequence and the auxRE containing a Myb binding sequence resulted in a significant reduction in expression levels as did a deletion involving the Myc and the third Myb binding sequences along with the second auxRE. However, no changes in expression patterns were observed. The results were quantified using transgenic root cultures.
Synthetic phosphopeptide from rhodopsin sequence induces retinal arrestin binding to photoactivated unphosphorylated rhodopsin
Authors: Puig Jaime , Arendt Anatol , Tomson Farol L. , Abdulaeva Galina , Miller Ron , Hargrave Paul A. , Hugh McDowell J.
Published: April 2003

A synthetic heptaphosphopeptide comprising the fully phosphorylated carboxyl terminal phosphorylation region of bovine rhodopsin, residues 330–348, was found to induce a conformational change in bovine arrestin. This caused an alteration of the pattern of limited proteolysis of arrestin similar to that induced by binding phosphorylated rhodopsin or heparin. Unlike heparin, the phosphopeptide also induced light‐activated binding of arrestin to both unphosphorylated rhodopsin in disk membranes as well as to endoproteinase Asp‐N‐treated rhodopsin (des 330–348). These findings suggest that one function of phosphorylation of rhodopsin is to activate arrestin which can then bind to other regions of the surface of the photoactivated rhodopsin.
Comparative proteolytic processing of rat prosomatostatin by the convertases PC1, PC2, furin, PACE4 and PC5 in constitutive and regulated secretory pathways
Authors: Brakch N. , Galanopoulou A.S. , Patel Y.C. , Boileau G. , Seidah N.G.
Published: April 2003

Recombinant vaccinia virus vectors were used to coexpress each of the candidate prohormone convertases PC1, PC2, furin, PACE4 and PC5 with rat prosomatostatin (rProSOM) in the constitutive secreting cell line LoVo and in the endocrine corticotroph cell line AtT‐20, which exhibits regulated secretion. Mammalian ProSOM is cleaved at a dibasic Arg‐Lys↓ site to produce somatostatin‐14 (S‐14) and at a monobasic Gln‐Arg↓ site to yield somatostatin‐28 (S‐28). The analysis of processed products by gel‐permeation high performance liquid chromatography shows that in LoVo cells PC1, furin and PACE4 generate S‐14, S‐28 and a mixture of S‐14 and S‐28, respectively, while PC2 is unable to process ProSOM in these constitutive cells. In contrast, PC2 can generate S‐14 in AtT‐20 cells. The convertase PC5 is unable to process ProSOM in either cell line. These data suggest that PC2, PC1 and PACE4 are candidate S‐14 convertases, while PACE4 and furin are candidate S‐28 convertases.
Improved measurement of13C,31P J coupling constants in isotopically labeled RNA
Authors: Legault Pascale , Jucker Fiona M. , Pardi Arthur
Published: April 2003

3JCPcoupling constants have been measured in 1 99%13C,15N labeled lead‐dependent ribozyme, known as the leadzyme. These coupling constants were determined by analysis of the intensity of individual crosspeaks in a spin‐echo difference constant time HSQC experiment. This procedure permits improved measurement of the3JC2′Pand3JC4′Pcoupling constants in isotopically labeled RNA and yielded valuable information on the β and ϵ backbone torsion angles in the leadzyme.
Iron release, membrane protein oxidation and erythrocyte ageing
Authors: Signorini C. , Ferrali M. , Ciccoli L. , Sugherini L. , Magnani A. , Comporti M.
Published: April 2003

The aerobic incubation of erythrocytes in phosphate buffer for 24–60 h (a model of rapid in vitro ageing) induced progressive iron release and methemoglobin formation. Membrane proteins showed electrophoretic alterations and increase in carbonyl groups (as documented by IR spectroscopy). None of these phenomena were seen when the erythrocytes were incubated under anaerobic conditions. The membranes from aerobically incubated cells bound a much higher amount of autologous IgG than those from anaerobically incubated ones, suggesting that the aerobic incubation gives rise to the senescent antigen. The addition of ferrozine during the aerobic incubation prevented both the IgG binding and the protein alterations seen in the IR spectra, suggesting an intracellular chelation of the released iron by ferrozine.
Reassembly of Synechocystis sp. PCC 6803 F1‐ATPase from its over‐expressed subunits
Authors: Steinemann D. , Engelbrecht S. , Lill H.
Published: April 2003

Subunits α, β, and γ of the F1‐part of cyanobacterial F0F1‐ATPase have been cloned into expression vectors. Overexpressed subunit β was found soluble in the cytoplasmic fraction of Escherichia coli cells under appropriate culture and induction conditions and was purified from cell extracts. Recombinant α and γ subunits precipitated into inclusion bodies and had to be solubilized, purified and refolded. The correct folding and functional integrity of the α and β subunits was monitored by their ability to bind nucleotides. Active cyanobacterial F1‐ATPase was assembled from its purified subunits α, β, γ, δ and ϵ. The reassembled enzyme reconstituted ATP synthesis in F1‐depleted thylakoid membranes of Synechocystis sp. PCC 6803 and hydrolyzed ATP.
Hepatocyte growth factor remains as an inactive single chain after partial hepatectomy or unilateral nephrectomy
Authors: Tang Wangxian , Miyazawa Keiji , Kitamura Naomi
Published: April 2003

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes and renal tubular epithelial cells. HGF is proteolytically activated in the tissue injured by hepatotoxin or nephrotoxin, suggesting that HGF functions as a crucial growth factor for tissue regeneration following hepatotoxin‐ or nephrotoxin‐induced injury. In this study, we analyzed the molecular form of HGF after partial hepatectomy or after unilateral nephrectomy. The active form of HGF was not detected under our experimental conditions after these operations. Thus, HGF may play little role in liver regeneration after partial hepatectomy and in compensatory renal enlargement after unilateral nephrectomy.
Selective inhibition of the mitochondrial permeability transition pore at the oxidation‐reduction sensitive dithiol by monobromobimane
Authors: Costantini Paola , Chernyak Boris V. , Petronilli Valeria , Bernardi Paolo
Published: April 2003

In this paper we introduce monobromobimane, a thiol reagent, as a selective blocker of the recently identified dithiol whose oxidation‐reduction status modifies voltage sensing by the mitochondrial permeability transition pore, a cyclosporin A‐sensitive channel. Monobromobimane does not inhibit the phosphate carrier, nor does it interfere with Ca2+ transport, energy coupling or ATP production and transport. We show that monobromobimane selectively prevents the shift in pore gating potential caused by some dithiol oxidants or crosslinkers but not by increasing [Ca2+], allowing a clear distinction of the pore agonists which act at this site.
The structure of porcine protegrin genes
Authors: Zhao Chengquan , Ganz Tomas , Lehrer Robert I.
Published: April 2003

We cloned the genes of three protegrins, a family of cathelin‐associated antimicrobial peptides originally isolated from porcine leukocytes. Each gene comprised 4 exons and 3 introns, wherein Exon I encoded the signal sequence and the first 37 amino acids of cathelin, Exons II and III contained 36 and 24 additional cathelin residues and Exon IV contained the final two cathelin residues followed by the protegrin sequence. This quadripartite gene structure helps explain how structurally diverse antimicrobial peptides can be expressed on common, cathelin‐containing precursors. Southern blot probed with an oligonucleotide specific for protegrin genes suggested that several identical or nearly identical protegrin genes were densely clustered in the pig chromosome.
cAMP‐dependent protein kinase activity is essential for preaggregative gene expression in Dictyostelium
Authors: Schulkes Conchita , Schaap Pauline
Published: April 2003

Constitutive inhibition of cAMP‐dependent protein kinase (PKA) in Dictyostelium cells blocks cell aggregation and development. We investigated the cause of the aggregation defect in transformants overexpressing dominant‐negative PKA regulatory subunits (PKA‐RM) under an actin 15 promoter. These mutants could not relay pulses of the chemoattractant cAMP, due to a defect in expression of the aggregative adenylyl cyclase (ACA) gene. Unstimulated and cAMP pulse‐induced expression of other aggregative genes encoding the cAMP receptor cAR1, adhesive contact sites A and cAMP‐phosphodiesterase were also strongly reduced in the mutants. Additionally, the expression of the discoidin I gene, that is expressed early in development in response to cell density sensing factors, was almost completely absent. These data are in interesting contrast with observations that cAMP relay and aggregative gene expression are normal in null mutants for the PKA catalytic (C) subunit and suggest the presence of multiple C subunit genes in Dictyostelium and an almost universal requirement for PKA activity in developmental gene expression.
ATP‐dependent efflux of calcein by the multidrug resistance protein (MRP): no inhibition by intracellular glutathione depletion
Authors: Feller N. , Broxterman H.J. , Währer D.C.R. , Pinedo H.M.
Published: April 2003

In this study we report that the multidrug resistance protein (MRP) transports calcein from the cytoplasmic compartment of tumor cells, in contrast to P‐glycoprotein which transports calcein acetoxymethyl ester from the plasmamembrane. The transport of calcein by MRP is ATP‐dependent and is inhibited by probenecid and vincristine. Intracellular glutathione (GSH) depletion which occurred when cells were exposed to buthionine sulfoximine had no effect on the efflux of calcein, whereas it reversed the daunorubicin accumulation deficit in MRP overexpressing tumor cells. In conclusion, ATP‐dependent transport of calcein and possibly other organic anions by MRP is not inhibited by a large decrease of the intracellular GSH concentration, that inhibits daunorubicin efflux by MRP.
Resistant P45051A1 activity in azole antifungal tolerant Cryptococcus neoformans from AIDS patients
Authors: Lamb D.C. , Corran A. , Baldwin B.C. , Kwon-Chung J. , Kelly S.L.
Published: April 2003

Azole antifungal compounds are important in the treatment of Cryptococcosis, a major cause of mortality in AIDS patients. The target of the azole drugs is P450 mediated sterol 14α‐demethylase. We have investigated the P450 system of Cryptococcus neoformans with respect to azole tolerance observed in clinical isolates which were obtained following the failure of fluconazole therapy. The clinical failure was correlated with in vitro tolerance of azole antifungal when compared to wild‐type strains. The microsomal P450 system was typical of yeast and fungi and fluconazole tolerance was not associated with defective sterol biosynthesis. The strains had slightly elevated P450 content and slightly reduced azole levels in the cells, but a clear cause for resistance was the increased level of drug needed to inhibit the sterol 14α‐demethylase in vitro.
Mutants of T7 RNA polymerase that are able to synthesize both RNA and DNA
Authors: Kostyuk D.A. , Dragan S.M. , Lyakhov D.L. , Rechinsky V.O. , Tunitskaya V.L. , Chernov B.K. , Kochetkov S.N.
Published: April 2003

A mutant T7 RNA polymerase (T7 RNAP) having two amino‐acid substitutions (Y639F and S641A) is altered in its specificity towards nucleotide substrates, but is not affected in the specificity of its interaction with promoter and terminator sequences. The mutant enzyme gains the ability to utilize dNTPs and catalyze RNA and DNA synthesis from circular supercoiled plasmid DNA. DNA synthesis can also be initiated from a single stranded template using a DNA primer. Another T7 RNAP mutant having only the single substitution S641A loses RNA polymerase activity but is able to synthesize DNA.
Interaction of rat liver lysosomes with basic polypeptides
Authors: Rukavishnikova Elena V. , Korolenko Tatjana A. , Sassa Toshihiro , Oka Tatsuzo , Horiuchi Saburou , Natoria Yasuo
Published: April 2003

In order to gain knowledge on the interaction of lysosomes with proteins, we have assessed the equilibrium densities of the lysosomal membrane and matrix markers after in vitro incubation of rat liver lysosomes with various polypeptides. The addition of basic polypeptides, polylysine or protamine, to the suspension of lysosomes brought about a profound alteration of lysosomal membrane, causing extensive leakage of lysosomal matrix enzymes. Electron microscopic observation revealed a remarkable aggregation of lysosomes by the basic polypeptides. Polyglutamic acid, an acidic polypeptide, did not produce such effect. ATP was found to stabilize lysosomes during incubation, particularly with basic polypeptides.
Antisense mapping the MOR‐1 opioid receptor: evidence for alternative splicing and a novel morphine‐6β‐glucuronide receptor
Authors: Rossi Grace C. , Pan Ying-Xin , Brown George P. , Pasternak Gavril W.
Published: April 2003

Although MOR‐1 encodes a mu opioid receptor, its relationship to the pharmacologically defined mu receptor subtypes has been unclear. Antisense mapping now suggests that these subtypes results from alternative splicing of MOR‐1. Three oligodeoxynucleotide probes targeting exon 1 and another oligodeoxynucleotide directed against the coding region of exon 4 block supraspinal morphine analgesia, a mu1 action, while five of six oligodeoxynucleotides directed against exons 2 and 3 are inactive. Inhibition of gastrointestinal transit and spinal morphine analgesia, two mu2 actions, are blocked only by the probe against exon 4 and not by those directed against exon 1. In contrast, the analgesic actions of the extraordinarily potent mu drug morphine‐6β‐glucuronide are blocked by six different antisense oligodeoxynucleotides targeting exons 2 and 3, but not by those acting on exons 1 or 4. These results suggest that the mu1 and mu2 receptor subtypes originally defined in binding and pharmacological studies result from alternative splicing of MOR‐1 while morphine‐6β‐glucuronide acts through a novel, previously unidentified receptor which is yet another MOR‐1 splice variant.
Cloning and characterization of a cDNA encoding a cellobiose dehydrogenase from the white rot fungus Phanerochaete chrysosporium
Authors: Raices Manuel , Paifer Edenia , Cremata José , Montesino Raquel , Ståhlberg Jerry , Divne Christina , Szabó István J , Henriksson Gunnar , Johansson Gunnar , Pettersson Göran
Published: April 2003

The cDNA of cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium has been cloned and sequenced. The 5′ end was obtained by PCR amplification. The cDNA contains 2310 translated bases excluding the poly(A) tail. The deduced mature protein contains 770 amino acid residues and is preceded by a 18 residue long signal peptide. The regions of the amino acid sequence corresponding to the heme and FAD domains of CDH were identified as well as the nucleotide‐binding motif, the disulfide pairing and a methionine residue chelating the heme iron. No homologous sequences were found for the heme domain, however, the FAD domain appears to be distantly related to the GMC oxidoreductase family.
Molecular mimicry and ankylosing spondylitis: possible role of a novel sequence in pullulanase of Klebsiella pneumoniae
Authors: Fielder M. , Pirt S.J. , Tarpey I. , Wilson C. , Cunningham P. , Ettelaie C. , Binder A. , Bansal S. , Ebringer A.
Published: April 2003

Molecular mimicry has been shown between two sequences of Klebsiella pneumoniae pulD secretion protein (DRDE) with HLA‐B27 (DRED) and pulA (pullulanase) enzyme (Gly‐X‐Pro) with types I, III and IV collagen respectively. IgG antibody levels in AS patients were elevated against 16mer synthetic peptides of HLA‐B27 and pulD by enzyme linked immunosorbent assay (ELISA) compared to controls (P < 0.001). ELISA assays against K. pneumoniae grown in the absence and presence of pullulan demonstrated significant levels of IgA antibody in AS patients compared to controls (P < 0.001). Increased IgA and IgG antibody levels to pulA and types I and IV collagen were observed in AS patients compared to controls (P < 0.001). These observations could be relevant in the sequence of molecular events in AS.