bluuebunny/crossref_metadata_2025 huggingface dataset:
Modification of cyanate ester resin by soluble polyarylates
Authors: Iijima Takao , Kunimi Takao , Oyama Toshiyuki , Tomoi Masao
Published: April 2003

Soluble polyarylates were prepared from the reaction of 2,2‐bis(4‐hydroxyphenyl)propane (bisphenol‐A) and aromatic acid dichlorides (phthaloyl chloride and related diacid dichlorides), and used to improve the brittleness of a cyanate ester resin. The polyarylates include poly[2,2‐di(4‐phenylene)propane phthalate] (PPA), poly[2,2‐di(4‐phenylene)propane phthalate‐co‐2,2‐di(4‐phenylene)propane isophthalate] (IPPA) and poly[2,2‐di(4‐phenylene)propane phthalate‐co‐2,2‐di(4‐phenylene)propane terephthalate] (TPPA). Furthermore, a commercial polyarylate, U‐polymer, was also used as a modifier. The morphologies of the modified resins depended on the polyarylate structure and concentration. The most effective modification of the cyanate ester resin could be attained because of the co‐continuous phase structure of the modified resin: 25 wt% inclusion of IPPA (50 mol% isophthalate units, weight average molecular weight (Mw) 38 500 g mol−1) led to a 130% increase in the fracture toughness (KIC) for the modified resin, with retention of its flexural properties and glass transition temperature, as compared with the values for the unmodified resin. Water absorptivity of the IPPA‐modified resin was smaller than that of the unmodified resin. Copyright © 2003 Society of Chemical Industry
Synthesis and reactivity of functionalized alkoxyamine initiators for nitroxide‐mediated radical polymerization of styrene
Authors: Li Irene Q , Knauss Daniel M , Priddy Duane B , Howell Bob A
Published: April 2003

The synthesis and examination of different functionalized (2,2,6,6‐tetramethyl‐1‐piperidinyloxy free radical) TEMPO‐containing alkoxyamine initiators for nitroxide‐mediated radical polymerization of styrene are reported. Initiators with ester and carbonate functional groups were synthesized by a low‐temperature radical‐abstraction reaction of the functionalized ethylbenzene in the presence of TEMPO to introduce the functional groups onto the initiating chain‐end of polystyrene. An initiator with two alkoxyamine groups symmetrically located at each end of a carbonate bond was also synthesized and used for nitroxide‐mediated styrene polymerization. Styrene polymerization using these initiators followed first‐order kinetics up to approximately 60 min at 140 °C or 30% monomer conversion. Alkoxyamines bearing an acetoxy or tert‐butylcarbonate group at the p‐position of 1‐(2,2,6,6‐tetramethyl‐1‐piperidinyloxy)ethylbenzene behave in a similar way to the unfunctionalized initiator. With an initiator containing two alkoxyamine groups, the resulting polymer molecular weight was twice that of the polymer obtained from initiators with only one alkoxyamine group, as expected from propagation from both chain‐ends. Upon hydrolysis of the carbonate bond, it was revealed that equivalent polymer chain growth occurred from each alkoxyamine site in the difunctional initiator. Copyright © 2003 Society of Chemical Industry
Stimuli‐responsive properties of aminophenylboronic acid‐carrying thermosensitive copolymers
Authors: Uğuzdoğan E , Kayı H , Denkbaş E B , Patır S , Tuncel A
Published: April 2003

Thermosensitive copolymers of N‐isopropylacrylamide (NIPA) and N‐acryloxysuccinimide (NASI) were obtained by solution polymerization using azobisisobutyronitrile as the initiator in a tetrahydrofuran–toluene mixture at 65 °C. A boronic acid‐carrying ligand, m‐aminophenylboronic acid (APBA) was covalently attached to the thermosensitive copolymer via the reaction between amino and succinimide groups. APBA‐coupled thermosensitive copolymer exhibited both temperature and pH sensitivity. Thermally reversible phase transitions were observed both in the acidic and alkaline pH region for the APBA‐modified copolymers obtained with different NASI feed concentrations. In our study, ribonucleic acid (RNA) was selected as a biomolecule having reactive groups which could potentially interact with the boronic acid functionality. The response of boronic acid‐carrying thermosensitive copolymer against RNA was investigated in aqueous media in the pH range 4–9. In the acidic pH region, an increase was observed in the lower critical solution temperature (LCST) of the APBA‐coupled thermosensitive copolymer with increasing RNA concentration. However, LCST decreased with increasing RNA concentration at both neutral and alkaline pH values. The LCST of the APBA‐attached copolymer varied linearly with the RNA concentration at pH of 3, 4 and 7.

© 2003 Society of Chemical Industry
Dispersion copolymerization of methyl methacrylate and acrylic acid in polar media: effects of reaction parameters on the particle size and size distribution of the copolymer microspheres
Authors: Huang Jin‐Xia , Yuan Xiao‐Ya , Yu Xiang‐Lin , Zhang Hong‐Tao
Published: April 2003

Micron‐size functional crosslinked poly(methyl methacrylate) (PMMA) particles with narrow size distribution in the range of 1∼5 µm were prepared by dispersion copolymerization in polar media with poly(N‐vinylpyrrolidone)(PVP) as steric stabilizer, 2,2′‐azobisisobutyronitrile(AIBN) as initiator and ethylene glycol dimethylacrylate (EGDMA) as crosslinking agent. The effects of functional comonomer acrylic acid (AA) concentration, contents in AIBN, EGDMA and PVP, media polarity as well as reaction temperature on the particle size and size distribution were investigated. Particle size initially increased, and then decreased with increasing AA concentration in the range of 0.7∼3.5 mol l−1, having a maximum of 5.01 µm at the concentration of 2.1 mol l−1, while size distribution became broader. This was regarded as the result of different roles of PAA in the process. Particle size increased with decreasing media polarity and stabilizer concentration, and with increasing initiator concentration and reaction temperature. The resulting particle shapes were observed by transmission electron microscopy and the presence of carboxyl groups on the surface of the particles was confirmed by Fourier‐transform infrared spectroscopy. Copyright © 2003 Society of Chemical Industry
Comparative study of the radiation‐induced grafting of styrene onto poly(tetrafluoroethylene‐co‐perfluoropropylvinyl ether) and polypropylene substrates. I: Kinetics and structural investigation
Authors: Cardona F , George GA , Hill DJT , Perera S
Published: April 2003

A comparative study has been made of the radiation grafting of styrene onto poly(tetrafluoroethylene‐co‐perfluoropropyl vinyl ether) (PFA) and polypropylene (PP) substrates, using the simultaneous irradiation method. Effects of grafting conditions such as monomer concentrations, type of solvent, dose rate and irradiation dose on the grafting yield were investigated. Under the same grafting conditions it was found that a higher degree of grafting of styrene was obtained using a mixture of dichloromethane/methanol solvents for PFA and methanol for PP and the degree of grafting was higher in PP than in PFA at all doses. However, the micro‐Raman spectroscopy analysis of the graft revealed that, for the same degree of grafting, the penetration depth of the grafted polystyrene into the substrate was higher in PFA than in PP substrates. In both polymers the crystallinity was hardly affected by the grafting process and the degree of crystallinity decreased slightly with grafting dose. The dependence of the initial rate of grafting on the dose rate and the monomer concentration was found to be 0.6 and 1.4 order for PFA and 0.15 and 2.2 for PP, respectively. The degree of grafting increased with increasing radiation dose in both polymers. However, the grafting yield decreased with an increase in the dose rate. The increase in the overall grafting yield for PFA and PP was accompanied by a proportional increase in the penetration depth of the graft into the substrates. Copyright © 2003 Society of Chemical Industry
Log‐normal distribution for a description of the molecular weight distribution of N‐acetylated chitosans depolymerized with hydrolytic enzyme
Authors: Shin‐ya Yoshitsune , Yoshizawa Toru , Hong Kyung‐Jin , Lee Moo‐Yeal , Kajiuchi Toshio
Published: April 2003

N‐acetylated chitosans (NACs) with different degrees of N‐acetylation (DAs) were enzymatically depolymerized at pH 5.1 and 40 °C, and the molecular weight distributions (MWDs) of the depolymerized NACs were then measured by size exclusion chromatography and were fitted by the log‐normal distribution function with two distribution parameters, β and M0. We discuss also the time‐evolution of the distribution parameters derived from the experimentally obtained MWD as well as the effects of experimental conditions, such as DA and initial NAC concentration (S0), on the distribution parameters. Copyright © 2003 Society of Chemical Industry
Syntheses and properties of polyurethanes containing side‐chain azobenzene groups
Authors: Kim Mi‐Ra , Kim Phan‐Seok , Kang Eun‐Hee , Ha Chang‐Sik , Lee Jin‐Kook
Published: April 2003

Polymers containing azobenzene groups have the characteristic reaction of photo‐induced cis–trans isomerism. The study of new materials for optical information storage has prompted making use of these photo‐isomerizations. In this study, we report the syntheses and properties of four different polyurethanes (DR‐PUns) containing azobenzene groups in the side‐chains. The structurally similar polyurethanes (DR‐PUns) were synthesized by the polycondensation reaction of Disperse Red 19 (DR 19) and four different diisocyanates in dimethylformamide. By introducing of DR 19 into the polymer, we obtained polymers containing a photochromic group in the side‐chain. The weight‐average molecular weights of the DR‐PUns were in the range 5500–12 900. The Tgs of the DR‐PUns range from 119.5 °C to 157.0 °C, depending on the structure of the diisocyanate. Optical properties and solubilities of the polyurethanes were compared. The diffraction efficiencies of films were measured as a function of the reaction time. Typically, the diffraction efficiencies of the DR‐PU1 film prepared from toluene 2,4‐diisocyanate were observed up to a level of 0.25%. For the DR‐PU1 film, the effect of the intensity of the induced laser beam on the diffraction efficiency is also discussed.

© 2003 Society of Chemical Industry
Swelling of acrylamide gels made at various onset temperatures: an optical transmission study
Authors: Pekcan Önder , Kara Selim
Published: April 2003

Optical transmission experiments were performed using a UV–visible spectrometer during the swelling of polyacrylamide (PAAm) gels. These gels were prepared from acrylamide with N,N′‐methylenebisacrylamide at various onset temperatures (Ton) by free radical crosslinking copolymerization in water and dried before use for swelling experiments. Transmitted light intensity (Itr) from the gels increased during early stages when PAAm gels were immersed in water at room temperature and then decreased continuously as swelling time was increased. Decrease in Itr was attributed to the increase in the scattered light intensity which may originate from the contrast between ‘frozen blob clusters’ and holes in the swelling gel. Decrease in Itr was modelled using the Li–Tanaka equation from which time constants (τ1) and collective diffusion coefficients (D0) were determined for the PAAm gels prepared at different onset temperatures. τ1 and D0 were found to be dependent on Ton. The correlation between Ton and τ1 and D0 was explained in terms of the size of microgels formed during gelation.

© 2003 Society of Chemical Industry
Erratum: Solution of periodic Poisson's equation and the Hartree‐Fock approach for solids with extended electron states: Application to linear augmented plane wave method, International Journal of Quantum Chemistry Volume 89, Number 2, 2002, pages 57–85
Authors: Nikolaev A.V. , Dyachkov P.N.
Published: April 2003

The original article to which this Erratum refers was published in Int J Quantum Chem 2002, 89, 57–85International Journal of Quantum Chemistry (2002) 89(2) 57–85
Dynamic Lie algebraic formulation of second‐order optical nonlinearity for substituted benzenes
Authors: Zhao Xian , Guan Daren , Yi Xizhang , Xu Guibao , Jiang Minhua
Published: April 2003

A dynamic Lie algebraic (DLA) formulation is applied to the study of nonlinear optical properties of the substituted benzenes. We have described the generation of the dynamic Lie algebra for the model Hamiltonian used in the present study. In terms of these elements of the dynamic algebra we express the evolution operator as a function of the group parameters, which can be determined by means of solving a system of coupled nonlinear differential equations. Thus, in terms of the density matrix operator formalism in statistical mechanics we obtain the statistical averages of the electric polarization and then derive readily an expression for the hyperpolarizability of the paradisubstituted benzenes. Comparisons with experimental observations and other quantum calculations of the hyperpolarizabilities for the paradisubstituted benzenes are made qualitatively and quantitatively. These results imply that the DLA method appears useful in describing the nonlinear optical phenomena in the substituted benzene molecules. © 2003 Wiley Periodicals, Inc. Int J Quantum Chem 93: 335–343, 2003
The KH module has an αβ fold
Authors: Castiglione Morelli Maria Antonietta , Stier Gunter , Gibson Toby , Joseph Catherine , Musco Giovanna , Pastore Annalisa , Travè Gilles
Published: April 2003

The KH module has recently been identified in a number of RNA associated proteins including vigilin and FMR1, a protein implicated in the fragile X syndrome. In this work, NMR spectroscopy was used to determine the secondary structure in solution of a KH domain (repeat 5 from vigilin). Almost complete assignments were obtained for the 1H and 15N resonances using uniform 15N‐labeling of the protein combined with homo‐nuclear 2D 1HNMR and 3D 15N correlated 1H NMR. On the basis of NOE patterns, secondary chemical shifts and amide solvent exposure, the secondary structure consists of an antiparallel three stranded β sheet connected by two helical regions. This domain may also be stabilized by an appended C‐terminal helix which is common to many but not all members of the KH family.
Alternative polyadenylation generates three low‐pI α‐amylase mRNAs with differential expression in barley
Authors: Skadsen Ronald W. , Knauer Nancy S.
Published: April 2003

Specific low‐pI α‐amylase genes from barley (Hordeum vulgare L.) produced alternative mRNAs with a 17‐base 3′ extension (extension 1) or a 17‐base extension beyond this (extension 2). The extended mRNAs do not arise from splicing of downstream sequences, and not all low‐pI genes contain the extended sequences. All three mRNAs occur in aleurones and shoots, while extension 2 is missing from scutella. Also, the unextended mRNAs predominate in total mRNA, but the extended mRNAs predominate in membrane‐bound polysomes. The extended sequences do not occur in previously characterized α‐amylases, but 16 of 18 bases, mainly in extension 1, are identical with a sequence in the 3′‐UTR of PAPI, a putative inhibitor of α‐amylase. These observations suggest that the extended sequences could play a functional role in α‐amylase expression.
Aspartate mutation distinguishes ETA but not ETB receptor subtype‐selective ligand binding while abolishing phospholipase C activation in both receptors
Authors: Rose Patricia M. , Krystek Stanley R. , Patel Pramathesh S. , Liu Eddie C.K. , Lynch Jean S. , Lach David A. , Fisher Susan M. , Webb Maria L.
Published: April 2003

The endothelin receptors, ETA and ETB, are G protein‐coupled receptors (GPCR) that show distinctively different binding profiles for the endothelin peptides and other ligands. We recently reported that Tyr129 in the second transmembrane region (TM2) of the ETA receptor was critical for subtype‐specific ligand binding [Krystek, S.R. et al. (1994) J. Biol. Chem. 269, 12383–12386]. Receptor models indicated that aspartic acids located one helical turn above (Asp133) and below (Asp126) Tyr129 in ETA had their side chains directed toward the putative binding cavity. Similarly in ETB, Asp147 and Asp154 are located one turn below and above His150, the residue that corresponds to Tyr129. Asp126 in ETA and Asp147 in ETB corresponds to the highly conserved aspartate present in TM2 of many GPCR that has frequently been shown to be crucial for agonist efficacy. Mutagenesis of Asp126 of the human ETA receptor to alanine resulted in an unaltered affinity for ET‐1, a 160‐fold increase in ET‐3 affinity and a decrease in affinity for the ETA selective naphthalenesulfonamide, BMS‐182874. ET‐1 activation of phospholipase C was abolished. In addition, despite the gain in binding affinity, ET‐3 failed to activate phospholipase C, suggesting that Asp126 is required for signal transduction. Mutagenesis of Asp133 to alanine indicated that it was critical only for the binding of BMS‐182874. In the ETB receptor, mutation of His150 to alanine or tyrosine indicated that it plays a minor role in ETB subtype‐selective ligand binding; mutation of the aspartates in TM2 of ETB did not alter ligand binding. As in the Asp126Ala ETA variant, ET‐1 and ET‐3 failed to increase intracellular levels of inositol phosphates in the Asp147Ala ETB mutant. Taken together, these data support the hypothesis that Asp126 and Asp133 flanking Tyr129 in TM2 of the ETA receptor play a role in defining ETA subtype‐selective ligand binding but Asp147 and Asp154 that flank the His150 in TM2 of the ETB receptor do not. Furthermore, these data indicate that Asp126 in ETA and Asp147 in ETB are important for transmembrane signaling via phospholipase C.
Mouse and human chromosomal assignments of mortalin, a novel member of the murine hsp70 family of proteins
Authors: Kaul Sunil C. , Wadhwa Renu , Matsuda Yoichi , Hensler Patrick J. , Pereira-Smith Olivia M. , Komatsu Yasuhiko , Mitsui Youji
Published: April 2003

Mortalin has been shown to exhibit differential distributions in cells with mortal and immortal phenotypes. In the present study, we report mot‐2 cDNA cloning from RS‐4 cells — an immortal clone from CD1‐ICR mouse embryonic fibroblasts — and the chromosomal assignments of mortalin related genes to mouse chromosomes 18 and X by fluorescence in situ hybridization. Similar analysis assigned the gene to chromosome 5q31.1 in human.
Retardation by the soybean Bowman‐Birk inhibitor of elastin hydrolysis catalyzed by leukocyte proteinases
Authors: Tikhonova Tamara V. , Gladysheva Inna P. , Larionova Natalia I.
Published: April 2003

The classical Bowman‐Birk inhibitor from soya retards strongly the hydrolysis of elastin catalyzed by leukocyte elastase, cathepsin G and a mixture of both. The inhibitory effect is practically unaffected by both the adsorption of the enzymes on elastin and prolongation of the enzymatic reaction.
Equatorial split of holo‐chaperonin from Thermus thermophilus by ATP and K+
Authors: Ishii Noriyuki , Taguchi Hideki , Sasabe Hiroyuki , Yoshida Masasuke
Published: April 2003

Holo‐chaperonin molecule from Thermus thermophilus is a bullet‐shaped particle whose cylinder part and round top are composed of two stacked rings of the cpn60 heptamer and a single ring of the cpn10 heptamer, respectively. We found that it splits at the plane between two cpn60 rings into two halves under physiological conditions, that is, in the presence of ATP (but not AMP‐PNP, ADP) + K+ (but not Na+) at 60°C. This equatorial split could be functionally important although it has not been considered in any current mechanistic model of chaperonin functioning.
Regulation and localization of midkine in rat ovary
Authors: Karino Satoshi , Minegishi Takashi , Ohyama Yoshio , Tano Mari , Nakamura Kazuto , Miyamoto Kaoru , Tanaka Shigeyasu , Ibuki Yoshito
Published: April 2003

In a previous experiment, it was shown that midkine (MK) was quite abundant in the follicular fluid; the concentration of MK in bovine follicular fluid was estimated to be 125 μg/l. To investigate the regulation of MK production in the ovary, we examined the effect of pregnant mare's serum gonadotropin (PMSG) and PMSG‐hCG treatment on the expression of MK in rat ovary. The mRNA of midkine (MK) was increased by PMSG injection and decreased by PMSG and hCG injection; the profile of change of mRNA of MK was similar to that of FSH receptor. Using in situ hybridization, we observed that the MK mRNA localized to granulosa cells. These results suggest that the granulosa cells produce MK under the control of gonadotropin.
H2‐forming N5,N10‐methylenetetrahydromethanopterin dehydrogenase: mechanism of H2 formation analyzed using hydrogen isotopes
Authors: Klein Andreas R. , Fernández Victor M. , Thauer Rudolf K.
Published: April 2003

H2‐forming N
5,N
10‐methylenetetrahydromethanopterin dehydrogenase catalyzes the reversible dehydrogenation of N
5,N
10‐methylenetetrahydromethanopterin (CH2 = H4MPT) to N
5,N
10‐methenyltetrahydromethanopterin (CH = H4MPT+) and H2. In D2O both HD and D2 are formed from CH2 = H4MPT and in H2O both HD and H2 from CD2 = H4MPT. Evidence is presented that HD is not an intermediate in the formation of D2 and H2, respectively.
Characterisation of the Sek‐1 receptor tyrosine kinase
Authors: Becker Nathalie , Gilardi-Hebenstreit Pascale , Seitanidou Tania , Wilkinson David , Charnay Patrick
Published: April 2003

We present an initial characterisation of the mouse Sek‐1 protein, a member of the Eph subfamily of putative receptor tyrosine kinases, which has been proposed to play a role in the segmentation of both the hindbrain and the mesoderm. Antibodies raised against the protein have been used to confirm the early embryonic expression pattern previously established by mRNA in situ hybridisation. These antibodies, together with the expression of the Sek‐1 gene in a baculovirus system, were instrumental in demonstrating that the protein carries a tyrosine kinase activity and that it is presented at the cell surface with its N‐terminal (putative ligand‐binding) domain outside of the cell. Therefore, as expected from its amino acid sequence, Sek‐1 conforms to the general model of receptor‐type tyrosine kinases.
ESR studies on the effect of ionic radii on displacement of Mn2+ bound to a soluble β‐galactoside binding hepatic lectin
Authors: Kayestha Rajeev , Sumati  , Hajela Krishnan
Published: April 2003

Binding of divalent metal ions to hepatic soluble β‐galactoside binding lectin was studied using electron spin resonance (ESR) spectroscopy. The Mn2+ bound to hepatic lectin could be displaced by Mg2+, Cu2+, Ni2+ and Ca2+ but not by Sr2+. As the ionic radii of Mg2+ (0.65 Å), Cu2+ (0.73 Å) and Ni2+ (0.72 Å) are appreciably smaller than Ca Mn2+ binding site is more accessible to Mg2+, Cu2+, and Ni2+ as compared to Ca2+, the ionic radius of Mn2+ being 0.80 Å. Sr2+ with an ionic radius of 1.13 is thus unable to displace bound Mn2+. Surprisingly, the presence of specific sugars like α‐lactose, or α‐d‐galactose facilitated the displacement of bound Mn2+ by metal ions whereas non‐specific sugars, i.e. α‐d‐glucose, β‐d‐fructose and α‐d‐ribose had no effect. It appears that minor perturbations in the saccharide binding site significantly affect the ability of the metal binding site to ligate bivalent metals.
Tendon extracellular matrix contains pentameric thrombospondin‐4 (TSP‐4)
Authors: Hauser Nik , Paulsson Mats , Kale Ashay A. , DiCesare Paul E.
Published: April 2003

In preparations of cartilage oligomeric matrix protein (COMP) from bovine tendon two contaminating polypeptides of 120 and 135 kDa were detected. N‐Terminal protein sequencing of these polypeptides showed homology to the N‐terminus and to an internal sequence in TSP‐4, respectively. TSP‐4 was further enriched by heparin affinity chromatography. Electron microscopy of this sample shows primarily five armed particles with globular domains at the periphery connected to a central assembly domain in which smaller N‐terminal globular domains can be resolved tightly packed at the center of the particle. We can thereby confirm the pentameric model for TSP‐4 proposed by Lawler et al. [(1995) J. Biol. Chem. 270, 2809–2814], on the basis of recombinantly expressed protein. We further show that TSP‐4 is abundant in tendon.
Oligomeric structure of a renal cystine transporter: implications in cystinuria
Authors: Wang Yan , Tate Suresh S.
Published: April 2003

Homologous proteins (NBAT) which mediate sodium‐independent transport of neutral as well as basic amino acids and cystine when expressed in Xenopus oocytes were recently cloned from mammalian kidneys. Mutations in human NBAT have been implicated in cystinuria. Here, we show that rat kidney and jejunal brush border membrane NBAT (85 kDa) is found in association with a 50 kDa protein. The association involves one or more interprotein disulfide bonds. Rabbit kidney brush border membranes and membranes of NBAT cRNA‐injected Xenopus oocytes also contain such heterodimers. Our data suggest that the heterodimer is the minimal functional unit of NBAT‐mediated amino acid transport and that the NBAT‐associated 50 kDa protein could play a role in cystinuria.
In vivo assembly of plasmid‐expressed ribosomal protein S7 of Thermus thermophilus into Escherichia coli ribosomes and conditions of its overexpression
Authors: Karginov Andrey V. , Karginova Olga A. , Spiridonova Vera A. , Kopylov Alexei M.
Published: April 2003

Researchers still have great difficulty in isolating individual ribosomal proteins from the ribosome in quantities high enough for structural research. To this end, when studying protein S7, we created an E. coli overproducer of the recombinant protein S7 of Thermus thermophilus. The vector for expression was pQE‐32 having a strong promoter of E. coli phage T5 and six triplets of His at the 5′‐end. This N‐terminal six His tag of the fusion protein is responsible for binding to Ni‐NTA‐resin and allows purifying the protein in one step. The yield of the recombinant protein was 20% and more of the total cellular proteins. In addition we have shown that the recombinant thermophilic protein is incorporated in vivo into the ribosome of E. coli despite the fact that these proteins (thermophilic and mesophilic) have a rather low homology, only 52%. This fact provides a base for the system to study functions of individual proteins.
Purification and structural characterization of the CD11b/CD18 integrin α subunit I domain reveals a folded conformation in solution
Authors: Fairbanks Michael B. , Pollock John R. , Prairie Mark D. , Scahill Terrence A. , Baczynskyj Lubo , Heinrikson Robert L. , Stockman Brian J.
Published: April 2003

The α subunits of the leukocyte CD11/CD18 integrins contain a ∼200 amino acid ‘inserted’ or I domain. The I domain of the cell‐surface Mac‐1(CD11b/CD18) integrin has been shown to be the major recognition site for several adhesion ligands, including iC3b, fibrinogen, factor X, and ICAM‐1. The I domain from the Mac‐1 α subunit has been expressed in Escherichia coli as a soluble GST‐fusion protein containing a factor Xa sensitive cleavage site. Analytical characterization of the purified I domain reveals that it is obtained in very high quality at high yields. CD and NMR spectra indicate that I domain adopts a predominantly folded structure in solution, independent of the remainder of the α subunit. Addition of Ca2+ and Mg2+ did not significantly perturb the structural conformation.
Gamma‐glutamyltransferase expression during all‐trans retinoic acid‐induced differentiation of hematopoietic cell lines
Authors: El yaagoubi Mohamed , Hachad Houda , Leh Hervé , Siest Gérard , Wellman Maria
Published: April 2003

Gamma‐glutamyltransferase activity, genes transcripts and differentiation by all‐trans retinoic acid have been investigated in cultured HL‐60, U937 and K562 cells. Acquisition of morphological and functional characteristics confirmed the terminal differentiation of HL‐60 and U937 cells. All‐trans r retinoic acid increased gamma‐glutamyltransferase activity in a cell type‐ and time‐dependent manner. Treatments with all‐trans retinoic acid isomers and structurally analogs showed that only retinoids with carboxylic acid group were able to induce enzyme activity in terminal differentiated cells. Additionally, the analysis of gamma‐glutamyltransferase genes transcription products demonstrated clearly that, both in untreated and in RA treated cells, only mRNA type I transcribed from the gene 6, was expressed.
Visualization of protein transport along the secretory pathway using green fluorescent protein
Authors: Kaether Christoph , Gerdes Hans-Hermann
Published: April 2003

We have expressed green fluorescent protein (GFP) from A. victoria in the secretory pathway of HeLa cells by fusing it to the C‐terminus of a secretory protein, chromogranin B. Under normal culture conditions at 37°C maturation of GFP to the fluorescent form was not detectable. However, fluorescent GFP was observed when biosynthetic protein transport was arrested at the intermediate compartment or the trans‐Golgi network by temperature blocks (15°C and 20°C, respectively). Reversal of the temperature blocks allowed the visualization of secretion of fluorescent GFP and offers the possibility to analyse transport in the secretory pathway in living cells.
Molecular cloning and sequence analysis of human preprocathepsin C
Authors: Pariš Alenka , Štrukelj Borut , Pungerčar Jože , Renko Metka , Dolenc Iztok , Turk Vito
Published: April 2003

A cDNA clone (C1) coding for human preprocathepsin C was isolated from a human ileum cDNA library using a rat kidney‐derived RT‐PCR probe and its complete nucleotide sequence determined. The full‐length 1857 bp sequence codes for a protein of 463 amino acid residues with a calculated molecular mass of 51848 Da. Comparison of the deduced amino acid sequence with that of rat preprocathepsin C indicates an 87.5% identity. A multiple alignment of the deduced cathepsin C sequence of 233 residues which, by analogy to other cysteine proteinases, corresponds to the mature protein, confirms that human cathepsin C belongs to the papain superfamily.
Abolition of G protein inhibition of α1A and α1B calcium channels by co‐expression of the β3 subunit
Authors: Roche John P. , Anantharam Vellareddy , Treistman Steven N.
Published: April 2003

Three different classes of α
1 Ca2+ channel (α
1A, α
1B, α
1C) were expressed in Xenopus oocytes to determine whether G protein‐mediated inhibition is an inherent property of the α
1, subunit itself, and if so, whether co‐expression of auxiliary subunits modulates the inhibition seen. From our data it is apparent that either α
1A or α
1B Ca2+ channels expressed alone are sufficient for voltage‐dependent G protein inhibition. α
1C Ca2+ channels expressed alone do not exhibit the G protein inhibition seen in α
1A and α
1B channels. Additionally, co‐expression of the β
3 subunit abolishes the ability of G proteins to inhibit currents through α
1A and α
1B Ca2+ channels. Differential sensitivity of α
1 as well as modulation of properties by β
3 provide a potential mechanism for the regulation of G protein‐mediated inhibition in neurons.
Stress‐induced tyrosine phosphorylation of actin in Dictyostelium cells and localization of the phosphorylation site to tyrosine‐53 adjacent to the DNase I binding loop
Authors: Jungbluth Andreas , Eckerskorn Christoph , Gerisch Günther , Lottspeich Friedrich , Stocker Susanne , Schweiger Anton
Published: April 2003

Actin is known to be phosphorylated at tyrosine, serine, or threonine residues in various cells. In cells of Dictyostelium discoideum, a rise in the tyrosine phosphorylation of actin is observed in response to ATP depletion. An actin fraction rich in phosphotyrosine was obtained by chromatography on the weak anion exchanger Mono‐P. Mass spectrometry and amino acid sequencing of protease cleavage products indicated that a single tyrosine residue was phosphorylated. Localization of this residue to position 53 of the actin sequence attributed the modification to a site that is critical for the capability of actin to polymerize. Induction of the tyrosine phosphorylation by heat shock and Cd2+ ions indicates that this modification of actin is implicated in the response of Dictyostelium cells to stress.
Site‐directed mutagenesis of Lys600 in phosphoenolpyruvate carboxylase of Flaveria trinervia: its roles in catalytic and regulatory functions
Authors: Gao Y. , Woo K.C.
Published: April 2003

Phosphoenolpyruvate carboxylases from various organisms contain two conserved lysine residues. In the C4 dicot Flaveria trinervia, one of these residues is Lys600. Converting this Lys600 to Arg600 or Thr600 mainly increased the K
m values and but had minimal effect on the V
max. The K
m for PEP, Mg2+ increased by up to 3‐fold in Arg600 and Thr600 but the K
m (HCO3
−) increased 9‐fold in Thr600, suggesting that Lys600 might be associated with bicarbonate‐binding. This lysine was not obligatory for enzyme activity although the wild‐type protein showed higher activity at physiological pH and was less inhibited by malate than the two mutants.
Amino acid residue: is it structural or functional?
Authors: Golovanov Alexander P. , Efremov Roman G. , Jaravine Victor A. , Vergoten Gérard , Arseniev Alexander S.
Published: April 2003

A new approach is suggested for delineating the structural and functional amino acid residues in proteins with known three‐dimensional structure, basing on the involvement of residues in intramolecular hydrophobic and hydrophilic interactions and additional information about the conservativity of the residues. The approach is applied to the families of homologous neurotoxins and cardiotoxins. The results obtained concerning the role of amino acid residues in both families of toxins accord well with the similarity of their fold, but different mechanisms of action. Current approach can be used for detailed characterization of protein spatial structures, as well as for rational protein engineering.
Involvement of pertussis toxin‐sensitive GTP‐binding proteins in sphingosine 1‐phosphate‐induced activation of phospholipase CCa2+ system in HL60 leukemia cells
Authors: Okajima Fumikazu , Tomura Hideaki , Sho Kimie , Nochi Hiromi , Tamoto Koichi , Kondo Yoichi
Published: April 2003

Exogenous sphingosine 1‐phosphate (S1P) induced Ca2+ mobilization, in association with an increase in inositol polyphosphate production reflecting activation of phospholipase C in HL60 leukemia cells. The increase in intracellular Ca2+ concentration ([Ca2+]i) induced by S1P was inhibited by an appropriate treatment of the cells with pertussis toxin (PTX), U73122 (a phospholipase C inhibitor) or phorbol 12‐myristate 13‐acetate (PMA). In parallel with the Ca2+ response, these agents also inhibited inositol polyphosphate production. The S1P‐induced Ca2+ response was also attenuated in the dibutyryl cAMP‐induced differentiated cells, where GTP‐binding protein‐induced Ca2+ response is suggested to be enhanced. Lysophosphatidic acid (LPA) also increased [Ca2+]i in the cells, but the maximal response was about half of that of S1P, and furthermore PTX and dibutyryl cAMP treatment hardly affected the LPA‐induced Ca2+ mobilization. We conclude that exogenous S1P mobilizes Ca2+ through phospholipase C activation. The S1P‐induced enzyme activation is at least partly mediated by PTX‐sensitive GTP‐binding protein‐coupled receptors which may be different from LPA receptors.
Identification of three amino acid residues in the B‐chain of platelet‐derived growth factor with different importance for binding to PDGF α‐ and β‐receptors
Authors: Kreysing Joachim , Östman Arne , van de Poll Monique , Bäckström Gudrun , Heldin Carl-Henrik
Published: April 2003

The B‐chain homodimer isoform of platelet‐derived growth factor (PDGF) binds with high affinity both to α‐ and to β‐receptors. In order to localize amino acid residues in PDGFBB of differential importance for the binding to the two receptors, PDGF‐BB mutants were analyzed in which single amino acid residues were changed to alanine residues. We found that Phe‐118 in loop 1 of the PDGF B‐chain is crucial for binding to both receptors, and that the surrounding amino acids, Asn‐117 and Leu‐119, appear to be important primarily for binding to the β‐receptor. In contrast, Lys‐161 in loop 3 was found to be more important for binding to α‐receptors than β‐receptors. Previous studies have shown that the receptor binding epitope of PDGF‐BB is composed mainly of loops 1 and 3; the findings of the present study show that the α‐ and β‐receptors interact with different amino acid residues in these regions.
Stable monomeric form of an originally dimeric serine proteinase inhibitor, ecotin, was constructed via site directed mutagenesis
Authors: Pál Gábor , Szilágyi László , Gráf László
Published: April 2003

Ecotin, a homodimer protein of E. coli, is a unique member of canonical serine proteinase inhibitors, since it is a potent agent against a variety of serine proteinases having different substrate specificity. Monomers of ecotin are held together mostly by their long C‐terminal strands that are arranged as a two‐stranded antiparallel β‐sheet in the functional dimer. One ecotin dimer can chelate two proteinase molecules, each of them bound to both subunits of ecotin at two different sites, namely the specific primary and the non‐specific secondary binding sites. In this study the genes of wild type ecotin and its Met84 Arg P1 site mutant were truncated resulting in new forms of ecotin that lack 10 amino acid residues at their C‐terminus. These mutants do not dimerize spontaneously, though in combination with trypsin they assemble into the familiar heterotetramer. Our data suggest that this heterotetramer exists even in extremely diluted solutions, and the interaction, which is responsible for the dimerization of ecotin, contributes to the stability of the heterotetrameric complex.
A 6 kDa protein homologous to the N‐terminus of the HMG1 protein promoting stimulation of murine erythroleukemia cell differentiation
Authors: Sparatore Bianca , Melloni Edon , Patrone Mauro , Passalacqua Mario , Pontremoli Sandro
Published: April 2003

Murine erythroleukemia (MEL) cells, in addition to an mRNA coding for a 30 kDa high mobility group (HMG)‐1 protein, contain an mRNA coding for a 6 kDa HMG1 protein having the following structural properties: (1) its primary structure has 90% homology with the N‐terminal sequence of the 30 kDa HMG1 protein; (2) it contains a consensus region of the HMG1 protein family; (3) it is deprived of the cluster of acidic amino acids that characterizes the C‐terminal region of the 30 kDa HMG1 protein. This novel small M
r HMG1 protein has been expressed in prokaryotic cells and tested to establish similarities and differences in activity compared to the homologous higher M
r HMG1 protein. It has been found that the low M
r HMG1 form is not released from MEL cells following induction to erythroid differentiation, but is still effective, although with much less efficiency, when added to the external medium, in promoting acceleration in the rate of MEL cell differentiation as well as in activation of α‐protein kinase C. Altogether these results provide evidence for the presence in MEL cells of a multigene family that encodes at least two different HMG1‐type sequences most presumably involved, at distinct cellular sites, in different functions although commonly related to the promotion of cell differentiation. Additional information can be considered concerning the relationship between the characteristic N‐terminal sequence of HMG1 protein and the extracellular activity on MEL cell differentiation.
Fractionation of carbon (13C/12C) isotopes in glycine decarboxylase reaction
Authors: Ivlev A.A. , Bykova N.V. , Igamberdiev A.U.
Published: April 2003

Fractionation of carbon isotopes (13C/12C) by glycine decarboxylase (GDC) was investigated in mitochondrial preparations isolated from photosynthetic tissues of different plants (Pisum, Medicago, Triticum, Hordeum, Spinacia, Brassica, Wolffia). 20 mM glycine was supplied to mitochondria, and the CO2 formed was absorbed and analyzed for isotopic content. CO2 evolved by mitochondria of Pisum was enriched up to 8% in 12C compared to the carboxylic atom of glycine. CO2 evolved by mitochondria of the other plants investigated was enriched by 5–16 % in 13C. Carbon isotope effects were sensitive to reaction conditions (pH and the presence of GDC cofactors). Theoretical treatment of the reaction mechanism enabled us to conclude that the value and even the sign of the carbon isotope effect in glycine decarboxylation depend on the contribution of the enzyme‐substrate binding step and of the decarboxylation step itself to the overall reaction rate. Therefore, the fractionation of carbon isotopes in GDC reaction was revealed which provides essential isotopic effects in plants in addition to the well‐known effect of carbon isotope fractionation by the central photosynthetic enzyme, ribulose‐1,5‐biphosphate carboxylase.
VDAC/porin is present in sarcoplasmic reticulum from skeletal muscle
Authors: Shoshan-Barmatz Varda , Hadad Nurith , Feng Wei , Shafir Iris , Orr Irit , Varsanyi Magdolna , Heilmeyer Ludwig M.G.
Published: April 2003

In this study we demonstrate the existence of a protein with properties of the voltage‐dependent anion channel (VDAC) in the sarcoplasmic reticulum (SR) using multiple approaches as summarized in the following: (a) 35 and 30 kDa proteins in different SR preparations, purified from other membranal systems by Ca2+/oxalate loading and sedimentation through 55% sucrose, cross‐react with four different VDAC monoclonal antibodies. (b) Amino acid sequences of three peptides derived from the SR 35 kDa protein are identical to the sequences present in VDAC1 isoform. (c) Similar to the mitochondrial VDAC, the SR protein is specifically labeled by [14C]DCCD. (d) Using a new method, a 35 kDa protein has been purified from SR and mitochondria with a higher yield for the SR. (e) Upon reconstitution into a planar lipid bilayer, the purified SR protein shows voltage‐dependent channel activity with properties similar to those of the purified mitochondrial VDAC or VDAC1/porin 31HL from human B lymphocytes, and its channel activity is completely inhibited by the anion transport inhibitor DIDS and about 80% by DCCD. We also demonstrate the translocation of ATP into the SR lumen and the phosphorylation of the luminal protein sarcalumenin by this ATP. Both ATP translocation and sarcalumenin phosphorylation are inhibited by DIDS, but not by atractyloside, a blocker of the ATP/ADP exchanger. These results indicate the existence of VDAC, thought to be located exclusively in mitochondria, in the SR of skeletal muscle, and its possible involvement in ATP transport. Together with recent studies on VDAC multicompartment location and its dynamic association with enzymes and channels, our findings suggest that VDAC deserves attention and consideration as a protein contributing to various cellular functions.
Human erythrocyte metabolism is modulated by the O2‐linked transition of hemoglobin
Authors: Messana Irene , Orlando Mario , Cassiano Loredana , Pennacchietti Lucia , Zuppi Cecilia , Castagnola Massimo , Giardina Bruno
Published: April 2003

The metabolic behaviour of human erythrocytes has been investigated with particular regard to the effect of their oxygenation state. Experiments performed at high phosphate concentration (80 mM) within the pH range 7.0–7.8 on erythrocytes at high (HOS) and low (LOS) oxygen saturation showed that at any pH value: (1) glucose consumption was independent of the oxygenation state; (2) pentose phosphate pathway (PPP) flux was about 2 times higher in the HOS than in the LOS state. At low phosphate concentration (1.0 mM) the PPP flux doubled in HOS as well as in LOS erythrocytes, whereas the decrease in glucose consumption was more marked in the HOS state. Metabolism of LOS erythrocytes approached that of HOS erythrocytes under the following conditions: (1) erythrocytes having band 3 modified by 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid; (2) CO‐saturated erythrocytes. These data support the hypothesis of a modulation of the relative rates of PPP and glycolysis achieved through competition between deoxy‐hemoglobin (deoxy‐Hb) and glycolytic enzymes for the cytoplasmic domain of band 3.
Structural and functional characterization of cytochrome c3 from D. desulfuricans ATCC 27774 by 1H‐NMR
Authors: Louro Ricardo O. , Pacheco Isabel , Turner David L. , LeGall Jean , Xavier António V.
Published: April 2003

Cooperativity between redox and protonation centres is known to be crucial for the function of complex proteins, but it is often difficult to describe in terms of thermodynamic parameters. Cytochrome c
3 is a good model for these studies since, while retaining the overall complexity of larger systems, it is suitable for detailed crystallographic and spectroscopic studies. Assignment of the haem substituent NMR resonances, together with NMR redox titrations of cytochrome c
3 from D. desulfuricans ATCC 27774, was used to correlate relative redox potentials to specific haems in the structure: haem . This order is different from that determined for the homologous proteins studied and in disagreement with that previously reported for this cytochrome (Morais, J., Palma, N., Frazão, C., Caldeira, J., LeGall, J., Moura, I., Moura, J.J.G. and Carrondo, M.A. (1995) Biochemistry 34, 12830–12841).
GTPase properties of the interferon‐induced human guanylate‐binding protein 2
Authors: Neun Rüdiger , Richter Marc F. , Staeheli Peter , Schwemmle Martin
Published: April 2003

Guanylate‐binding proteins (GBPs) were originally described as proteins that are strongly induced by interferons and are capable of binding to agarose‐immobilized guanine nucleotides. hGBP1, the first of two members of this protein family in humans, was recently shown to represent a novel type of GTPase that hydrolyzes GTP predominantly to GMP. We now report that purified recombinant hGBP2 also hydrolyzes GTP very efficiently, although GDP rather than GMP was the major reaction product. The biochemical parameters of this reaction were as follows: K
m=313 μM, turnover number = 22 min−1. Both hGBP1 and hGBP2 failed to hydrolyze GDP, however, GDP was an effective inhibitor of the hGBP2‐ but not the hGBP1‐catalyzed GTP hydrolysis reaction. Thus, hGBP1 and hGBP2 have similar biochemical properties, but show pronounced differences in product specificity.
Cloning of a novel ubiquitin‐conjugating enzyme (E2) gene from the ciliate Paramecium tetraurelia
Authors: Okano Satoshi , Tokushima Hideyuki , Nakaoka Yasuo , Shimizu Kikuo
Published: April 2003

We isolated a 1.7 kb gene (UbcPl) for a ubiquitinconjugating enzyme from a P. tetraurelia cDNA library and sequenced it. Its deduced polypeptide sequence consists of 425 amino acid residues (48 kDa). The UbcP1 protein contains novel N‐ and C‐terminal extensions in addition to a UBC domain, and within the UBC domain it shares low identity with sequences of other known E2s. A constructed phylogenetic tree suggests that the UbcP1 protein may represent a member of a distinct subfamily of E2s. Southern blot analysis showed that the N‐terminal extension of the UbcP1 is conserved in P. multimicronucleatum.