bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a rat liver cDNA and mRNA coding for the 28 kDa gap junction protein
Authors: Heynkes Roland , Kozjek Gisela , Traub Otto , Willecke Klaus
Published: July 2002

By screening of a rat liver cDNA library with complex and deoxyinosine containing oligonucleotide probes a cDNA clone was isolated and shown by sequencing to code for the amino‐terminal half of the rat liver 28 kDa gap junction protein. The insert hybridized to a 1.9 kb species from rat and mouse liver poly(A)+ RNA in Northern blot analysis. In embryonic mouse hepatocytes the amount of the 1.9 kb mRNA increased 3‐fold between 24 and 96 h in culture. This correlates with the previously described increase of the 28 kDa gap junction protein under these conditions.
Salt dependent dimerisation of caldesmon
Authors: Cross R.A. , Cross K.E. , Small J.V.
Published: July 2002

Using analytical gel filtration (FPLC) we show here that avian gizzard caldesmon (chain molecular mass 150 kDa) self‐associates to form end‐to‐end dimers. Increasing salt concentration promotes dimerisation: at 150 mM KCl, about 40% of the caldesmon was dimeric. Freshly gel filtered caldesmon had an actin gelating activity which decreased with increasing ionic strength. At 150 mM KCl, caldesmon at a 1:90 molar ratio to actin doubled the low shear viscosity of F‐actin. Sixfold less filamin was required to produce the same effect.
Regiospecific oxygenations during ring cleavage of a secondary metabolite, 3,4‐dimethoxybenzyl alcohol catalysed by lignin peroxidase
Authors: Shimada M. , Hattori T. , Umezawa T. , Higuchi T. , Uzura K.
Published: July 2002

Enzymatic oxidation of veratryl alcohol yielded a new ring cleavage product (δ‐lactone) in addition to the two known γ‐lactone products. The experiment with 18O‐enriched water and dioxygen clearly showed that one oxygen atom each from water and dioxygen is specifically incorporated into the cleavage product at the original C3 or C4 position of 3,4‐dimethoxybenzyl alcohol. A new type of reaction mechanism proposed for the ring cleavage of this compound is rationally explained in good accord with the one‐electron transfer mechanism.
Substitution of phosphatidylserine by lipid A in the activation of purified rabbit brain protein kinase C
Authors: Ellis Christine A. , Aitken Alastair , Takayama Kuni , Qureshi Nilofer
Published: July 2002

Three lipid A derivatives (hexaacyl monophosphoryl lipid A, hexaacyl diphosphoryl lipid A, and disaccharide precursor IV A) were shown to activate protein kinase C from rabbit brain. These derivatives substituted for phosphatidylserine in a concentration‐dependent manner and did not compete for binding of [3H]phorbol dibutyrate to its receptor site. Instead, phorbol dibutyrate binding was increased on raising the concentration of the derivatives in a similar manner to phosphatidylserine. The phorbol ester 12‐0‐tetra‐decanol 13‐acetate augmented the activation of protein kinase C by the lipid A derivatives.
Molecular cloning and expression of Clostridium difficile toxin A in Escherichia coli K 12
Authors: Wren Brendan W. , Clayton Christopher L. , Mullany Peter P. , Tabaqchali Soad
Published: July 2002

Clostridium difficile toxin A was purified to homogeneity and was used to raise monospecific antiserum in rabbits. A gene bank of C. difficile DNA in Escherichia coli was constructed by cloning Sau3A‐cleaved clostridial DNA fragments into the bacteriophage vector λEMBL3. Out of 4500 plaques screened with antitoxin A, 9 clones were positively identified. One of these clones λtA5 expressed a 235 kDa protein which exhibited a cytotonic effect on Chinese hamster ovary cells, and had the ability to haemagglutinate rabbit erythrocytes, both properties characteristic of toxin A. The size of the λtA5 insert DNA was 14.3 kb.
Phorbol ester induces loss of VIP stimulation of adenylate cyclase and VIP‐binding sites in HT29 cells
Authors: Bozou Jean-Claude , Couvineau Alain , Rouyer-Fessard Christiane , Laburthe Marc , Vincent Jean-Pierre , Kitabgi Patrick
Published: July 2002

Treatment of HT29 cells with the tumor promoting phorbol ester PMA resulted in an attenuation of VIP‐stimulated cAMP production in intact cells and VIP‐stimulated adenylate cyclase activity in cell membranes. PMA did not decrease the ability of cholera toxin and forskolin to elevate cAMP levels in intact cells. Fluoride‐stimulated adenylate cyclase activity in HT29 cells homogenates was not affected byPMA. The maximal VIP binding capacity of homogenates prepared from HT29 cells treated with PMA was decreased by 50%. It is concluded that protein kinase C regulates VIP receptor function possibly through phosphorylation of the VIP receptor.
Structure determination of the O‐specific polysaccharides from Citrobacter O4‐ and O27‐lipopolysaccharides by methylation analysis and one‐ and two‐dimensional 1 H‐NMR spectroscopy
Authors: Romanowska Elżbieta , Romanowska Anna , Dabrowski Janusz , Hauck Michael
Published: July 2002

Using sugar and methylation analyses, and one‐ and two‐dimensional 1H‐NMR spectroscopy at 500 MHz it was established that poly‐β‐1,2‐4‐deoxy‐D‐arabinohexopyranose occurs as O‐specific chains of lipopolysaccharides in Citrobacter serotypes O4, O27 and O36. Strong serological cross‐reactivity between these serotypes is in full agreement with the chemical identity of their O‐specific polysaccharides.
Sequence identity between a lysine‐containing peptide from Leuconostoc mesenteroides glucose‐6‐phosphate dehydrogenase and an active site peptide from human erythrocyte glucose‐6‐phosphate dehydrogenase
Authors: Bhadbhade Mohan M. , Adams Margaret J. , Flynno T.Geoffrey , Richard Levy H.
Published: July 2002

Peptides recently isolated and sequenced from a bacterial (Leuconostoc mesenteroides) glucose‐6‐phosphate dehydrogenase are remarkably homologous to an active site region of the human erythrocyte enzyme, although the enzymes differ in their overall amino acid composition and kinetic properties. The computer program ALIGN, used to determine the bestalignment between the two enzyme sequences, gives matchscores which are statistically highly significant.
Identification of zymogen and mature forms of human carboxypeptidase H A processing enzyme for the synthesis of peptide hormones
Authors: Hook Vivian Y.H. , Affolter Hans-Urs
Published: July 2002

Carboxypeptidase H (CPH) is one of several processing enzymes required for the conversion of peptide hormone precursors into their smaller active forms. In this study, high levels of CPH activity was found in a liver metastasis of a human ideal carcinoid which expresses β‐preprotachykinin mRNA and the tachykinin neuropeptides, substance P and substance K. This human CPH showed properties of a zinc‐metallopeptidase that is structurally similar to bovine and rat CPH. Immunoblots of the human ileal carcinoma with anti‐bovine CPH showed that CPH activity is represented by two proteins of apparent molecular masses 57 and 55 kDa. Cell‐free translation of poly(A)+ RNA followed by immunoprecipitation with anti‐bovine CPH showed that human CPH mRNA encodes a precursor protein of apparent molecular mass 75 kDa. These data demonstrate that human CPH is synthesized as a zymogen, prepro‐CPH, which must be cleaved to form catalytically active CPH.
Importance of folded monomer and extended antiparallel dimer structures as enkephalin active conformation Molecular dynamics simulations of [Met5]enkephalin in water
Authors: Yoneda Shigetaka , Kitamura Kunihiro , Doi Mitsunobu , Inoue Masatoshi , Ishida Toshimasa
Published: July 2002

Simulations of the molecular dynamics of the [Met5]enkephalin monomer and dimer structures in water have been carried out. The dynamic trajectories have been analyzed in terms of the distances between intra‐ or intermolecular polar atoms. The time‐correlated conformational transitions of an extended monomer structure have been converged into a stationary state among the β‐bend folded forms. However, the dynamics simulation of an extended antiparallel dimer structure has shown no noticeable conformation change. These results imply that both the β‐bend monomer and the extended dimer structures exist together as the fundamental conformation of enkephalins.
Evidence for phosphorylation of yeast phosphofructokinase
Authors: Huse Klaus , Jergil Bengt , Schwidop Wolf-Dieter , Kopperschläger Gerhard
Published: July 2002

Radioactively labelled material from yeast cells grown in the presence of [32P]phosphate was specifically recognized by antibodies raised against yeast phosphofructokinase. Purified yeast phosphofructokinase was phosphorylated in a cyclic AMP‐independent manner by a protein kinase enriched from yeast extracts. This phosphorylation occurred specifically on the β‐subunit, and 0.56 mol of phosphate/mol of subunit was incorporated. The results indicate the phosphorylation of yeast phosphofructokinase both in vivo and in vitro. Phosphofructokinase phosphorylated in vitro was more stable against proteolytic degradation compared to the non‐phosphorylated enzyme.
Structure of the ATP‐synthase studied by electron microscopy and image processing
Authors: Tsuprun Vladimir L. , Orlova Elena V. , Mesyanzhinova Irina V.
Published: July 2002

The structure of ATP synthase from beef heart mitochondria has been studied by electron microscopy and image processing using negatively stained specimens of detergent‐solubilized and membrane‐bound molecules. The F1‐ATPase and the membrane‐embedded F0 sector of ATP synthase are found to be connected by a narrow stalk, 4–4.5 nm long and 3–3.5 nm wide, projecting about 4.2 nm from the membrane surface. The F0 sector has a globular shape, 6–8 nm in diameter and, in part, extends from the membrane lipid bilayer.
Assignment of imidazole resonances from two‐dimensional proton NMR spectra of bovine Cu,Zn Superoxide dismutase
Authors: Paci Maurizio , Desideri Alessandro , Sette Marco , Ciriolo Maria R. , Rotilio Giuseppe
Published: July 2002

Two‐dimensional 1H‐NMR spectra were carried out on bovine Cu(I),Zn Superoxide dismutase. The ring protons of the single tyrosine and of the 4 phenylalanines were identified from COSY spectra. Prom NOESY spectra all imidazole C‐resonances could be specifically assigned to each of the 8 histidines using the crystal coordinates of the Cu(II),Zn enzyme. Since 6 histidines are involved in the structure of the active site, this result implies nearly identical active site conformations for the two oxidation states of the catalytic cycle of this enzyme, in line with its diffusion‐limited rate.
Secretion of recombinant ribonuclease T1 into the periplasmic space of Escherichia coli with the aid of the signal peptide of alkaline phosphatase
Authors: Fujimura Takao , Tanaka Toshiki , Ohara Kanako , Morioka Hiroshi , Uesugi Seiichi , Ikehara Mono , Nishikawa Satoshi
Published: July 2002

The ribonuclease T1 (RNase T1) gene was ligated to a synthetic gene for the signal peptide of Escherichia coli alkaline phosphatase. When this fusion gene was expressed in E.Coli under the control of the trp promoter, active RNase T1 having the correct N‐terminal sequence was secreted into the periplasmic space, indicating that the heterologous signal peptide had been cleaved off correctly. The enzyme could be readily purified from the periplasmic fraction with a yield of 1.8 mg from 1 liter culture. Adopting the same strategy, it was possible to produce a labile mutant of RNase T1 (Glu‐58 → Ala mutant) in E. coli, the yield of the purified mutant enzyme being 2.0 mg from 1 liter culture.
Isolation of allophycocyanin B from Rhodella violacea results in a model of the core from hemidiscoidal phycobilisomes of rhodophyceae
Authors: Reuter Wolfgang , Nickel Claudia , Wehrmeyer Werner
Published: July 2002

Two ‘trimeric’ allophycocyanin complexes could be isolated from the hemidiscoidal phycobilisomes of Rhodella violacea, AP = (α∗Apα2
APβ2
APβ∗AP) and APB = (α∗ApαAPαAPBβ2
APβ∗AP) · Lc
13.5APB. The isolation was performed by combined methods of gradient centrifugation, hydroxylapatite chromatography and ‘native’ polyacrylamide gel electrophoresis. AP showed the well‐known spectral characteristics of allophycocyanin without linker polypeptide. APB is characterized by its long wavelength absorbing shoulder (675 nm) and fluorescence emission (682nm), respectively. The existence of two low molecular linker polypeptides Lc
12.5 and Lc
13.5APB in the phycobilisomes of Rhodella violacea, their stoichiometric calculations and the localization of Lc
13.5APB in allophycocyanin B facilitated the construction of a model of the phycobilisome core.
Complete localization of the disulfide bridges and glycosylation sites in boar sperm acrosin
Authors: Töpfer-Petersen E. , Calvete J. , Schäfer W. , Henschen A.
Published: July 2002

Acrosin is a disulfide‐bonded two‐chain glycoprotein, which belongs to the serine proteinase family and which plays a central role in mammalian fertilization. The amino acid sequence of acrosin from different species has been recently derived by cDNA analysis. Boar sperm acrosin contains twelve cysteine residues forming two interchain and 4 intrachain disulfide bonds. Protein‐chemical and mass‐spectroscopic analyses of fragments and subfragments obtained by proteolytic and chemical degradation of the isolated protein allowed the unambiguous localization of all disulfide bridges and glycosylation points in boar acrosin. The 12 cysteines and the glycosylated asparagines in the porcine enzyme are absolutely conserved in number and position within all known acrosin sequences. Thus, the disulfide bond and glycosylation patterns outlined here are conserved during evolution and may be important for enzyme function.
Protection of reaction center II from photodamage by low temperature and anaerobiosis in spinach chloroplasts
Authors: Kirilovsky Diana , Etienne Anne-Lisc
Published: July 2002

The rate of the irreversible damage to the reaction center II, caused by exposure of spinach thylakoids to high light was slowed down by anacroble conditions and by lowering the temperature. The protective mechanisms of these conditions were different. In both cases F
max decreased more slowly than in control photoinhibition. A reversible intermediate step was only observed under unacrobic conditions. This state was inactive for oxygen evolution and it was characterized by an increase of F
v.
Mutations in a semiconserved region of the Tetrahymena intron
Authors: Pace Umberto , Szostak Jack W.
Published: July 2002

The A‐rich in paired region P5a of the Tetrahymena intron is a structural feature that is conserved in the sub‐group Ib self‐splicing introns. We have constructed a series of substitution and deletion mutations in this region of the intron. Kinetic analysis has shown that some of the mutanis have a reduced maximal extent of splicing, while others have a reduced V
max. These mutations could be reactivated to a great extent by spermidine and high Mg2+ concentrations. These data are consistent with the hypothesis that the A‐rich bulge of P5a has a role in stabilizing the higher‐level structure of the ribozyme.
Purification and functional characterization of the human β2‐adrenergic receptor produced in baculovirus‐infected insect cells
Authors: Reiländer Helmut , Boege Fritz , Vasudevan Subhash , Maul Gabi , Hekman Mirko , Dees Christian , Hampe Wolfgang , Helmreich Ernst J.M. , Michel Hartmut
Published: July 2002

A human cDNA fragment bearing the complete coding region for the β2‐adrenergic receptor was introduced into the genome of Autographa california nuclear polyhedrosis virus under the control of the polyhedrin promoter. Binding studies using [125I]iodocynnopindolol showed that Sf9 insect cells infected with the recombinant virus expressed ≈ 1 × 104 β2‐adrenergic receptors on their cell surface. Photoaffinity labeling of whole cells and membraines revealed a molecular weight of ≈ 46000 for the expressed receptor. The receptor produced in insect cells is glycosylated but the extent and pattern differ from that of the receptor from human tissue. The heterologously expressed receptor was purified by alprenolol affinity chromatography, and was able to activate isolated Gs‐protein.
Inhibition of protein kinase C arrests proliferation of human tumors
Authors: Miñana Maria-Dolores , Felipo Vicente , Cortés Francisco , Grisolía Santiago
Published: July 2002

We have shown that inhibition of protein kinase C by 1‐5‐isoquinolinylsulfonyl‐2‐methylpiperazine, H7, induces differentiation and inhibits proliferation of Neuro 2a cells. We have now tested if H7 is able to inhibit proliferation of: 1) human tumor cell lines from tissues other than brain; and 2) primary cultured cells from several human brain tumors. H7 inhibits, in a dose‐dependent manner, proliferation of all human tumor cell lines tested and of primary cultured cells from human brain tumors. These results indicate that inhibition of protein kinase C inhibits proliferation of tumoral cells, therefore, H7, and likely other inhibitors of protein kinase C, could be useful in the clinical treatment of brain (and probably other) tumors.
Glycogen hyperaccumulation in Saccharomyces cerevisiae ras2 mutant A biochemical study
Authors: Fernández-Bañares Isabel , Clotet Josep , Ariño Joaquin , Guinovart Joan J.
Published: July 2002

The mechanism by which yeast ras2 mutant hyperaccumulates glycogen has been investigated. Total glycogen synthase activity was between and 1.3 times higher in the ras2 mutant than in an isogenic strain. In addition, while in the normal strain the glycogen synthase activation state decreased along the exponential phase, in the mutant strain the opposite behaviour was observed: glycogen synthase activation state rose continuously reaching full activation at the beginning of the stationary phase. Glycogen phosphorylase a activity was up to 40 times higher in the mutant than in the normal strain. Glucose 6‐phosphate and fructose 2,6‐bisphosphate levels were slightly more elevated in the mutants. The increase in total glycogen synthase and, particularly, the full activation of this enzyme may explain glycogen hyperaccumulation in the ras2 mutant even in the presence of elevated levels of glycogen phosphorylase a.
Expression in animal cells of the 5‐HT1A receptor by a vaccinia virus vector system
Authors: Ho Begonia Y. , Karschin Andreas , Raymond John R. , Branchek Theresa , Lester Henry A. , Davidson Norman
Published: July 2002

The co‐infection or infection—transfection variants of the T7 RNA polymerase/vaccinia vector system were used to express 5‐HT1ARs in COS‐7, BSC‐40 and GH3 cells, with co‐infection giving ca. 3‐fold higher level than infection—transfection. Binding affinities were similar to those of the endogenous 5‐HT1AR, with highest affinities for 5‐HT and 8‐OH‐DPAT. Functional properties were demonstrated by assays of agonist‐stimulated GTPase activity and its inhibition by pertussin toxin. Immunoblot assays showed expression of the unglycosylated and glycosylated receptor protein in the membrane and, surprisingly, in the cytosolic fractions.
The gene structure of tetranectin, a plasminogen binding protein
Authors: Berglund Lars , Petersen Torben E.
Published: July 2002

The gene for human tetranectin was isolated from a genomic library with a mixture of degenerate oligonucleotide probes. The gene is about 12 kbp and contains two intervening sequences. The gene encodes a protein of 202 amino acid residues, with a signal peptide of 21 amino acid residues, followed by the tetranectin sequence of 181 amino acid residues. Northern blot analysis revealed that tetranectin mRNA was present in all eight tissues tested with the highest concentration in lung. Southern blot analysis showed hybridization to two genes. Further investigations are needed to determine whether the genes are allelic or non‐allelic.
Oxidation of methylamine by a Paracoccus denitrificans mutant impaired in the synthesis of the bc1 complex and the aa3‐type oxidase Evidence for the existence of an alternative cytochrome c oxidase in this bacterium
Authors: De Gier Jan-Willem L. , Van Spanning Rob J.M. , Oltmann L.Fred , Stouthamer Adriaan H.
Published: July 2002

A Paracoccus denitrificans fbcC—ctaDII double mutant strain impaired in the synthesis of both the bc
1 complex and the aa
3‐type oxidase has been constructed. This mutant strain, which is still able to grown on methylamine as sole carbon and energy source, exhibits unimpaired oxygen consumption with succinate, methylamine and endogenous substrates as electron donors. From kinetic studies of the oxidation and reduction rates of cytochromes c, it can be concluded that P. denitrificans contains a second cytochrome c oxidase, different from the aa
3‐type.
Phosphorylation of GDI and membrane cycling of rab proteins
Authors: Steele-Mortimer Olivia , Gruenberg Jean , Clague Michael J.
Published: July 2002

Membrane transport is known to be regulated by protein phosphorylation and by small GTPases of the rab family. Using specific antibodies, we have identified a 55 kDa phosphorylated protein which co‐immunoprecipitated with the cytosolic forms of rab5 and other rab proteins. We demonstrate, on the basis of its mobility in two‐dimensional electrophoresis gels and its immunological properties, that this protein is rab GDI (p55/GDI). We also found that, a minor fraction of p55/GDI is membrane associated, but, whilst also complexed with rab proteins, it is not phosphorylated. On the basis of these data we suggest that the cycling of rab proteins between membranes and cytosol is regulated by phosphorylation of p55/GDI.
Molecular structure of ras‐related small GTP‐binding protein genes of rice plants and GTPase activities of gene products in Escherichia coli
Authors: Kidou Shin-ichiro , Anai Toyoaki , Umeda Masaaki , Aotsuka Satoshi , Tsuge Tomohiko , Kato Atsushi , Uchimiya Hirofumi
Published: July 2002

We isolated two rice cDNA clones (rid and ric2) encoding proteins homologous to the ras‐related small GTP‐binding protein. The amino acid sequences of rid and ric2 are conserved in four regions involved in GTP binding and hydrolysis which are characteristic in the ras and ras‐related small GTP‐binding protein genes. In addition, two consecutive cysteine residues near the carboxyl‐terminal end required for membrane anchoring are also present in rid and ric2. The rid and ric2 proteins synthesized in Escherichia coli possessed GTPase activity (i.e. hydrolysis of GTP to GDP).
A novel, specific pro‐urokinase complex on monocyte‐like cells, detected by transglutaminase‐catalyzed cross‐linking
Authors: Behrendt Niels , Rønne Ebbe , Danø Keld
Published: July 2002

Radiolabeled pro‐urokinase plasminogen activator (pro‐uPA) was cross‐linked to a specific protein on the surface of human monocyte‐like U937 cells in a reaction catalyzed by tissue transglutaminase. The conjugate formed with this unknown component had a much higher molecular weight (apparent M
r, 250,000–300,000) than the complex of pro‐uPA and the urokinase plasminogen activator receptor (uPAR). There was a strong preference for the pro‐form of uPA. The conjugate was recognized by antibodies against uPA but not by anti‐uPAR antibodies. Nevertheless, the blocking of uPAR with a monoclonal antibody abolished the formation of the conjugate, thus showing a role of uPAR in this process.
Molecular characterization of a blue visual pigment gene in the fish Astyanax fasciatus
Authors: Yokoyama Ruth , Yokoyama Shozo
Published: July 2002

We report here the isolation and sequence determination of a gene closely linked to the Astyanax red visual pigment gene. Reverse transcription polymerase chain reaction assays show that this new gene (B23Aƒ
) and the previously characterized red and green visual pigment genes of Astyanax are all expressed in the eye. Phylogenetic analysis shows that B23Aƒ
belongs to the group consisting of short wavelength‐sensitive pigment genes from different species and is most closely related to the goldfish blue visual pigment gene.
Cloning and sequence analysis of the gene encoding the low potential cytochrome c of Synechocystis PCC 6803
Authors: Kang Charmaine , Chitnis Parag R. , Smith Stanley , Krogmann David W.
Published: July 2002

The gene for the low potential cytochrome c (petk) was isolated from a genomic library of Synechocystis PCC 6803. The nucleotide sequence of this gene contains two regions with sequence similarity to two regions in the gene for the high potential cytochrome c
6 of the organism. The sixth iron ligand can be identified with a conserved histidine. Experiments demonstrate the reduction of the low potential cytochrome by reduced ferredoxin II. The heme of the cytochrome is flanked by lysines which may be involved in orienting the ferredoxin near the site of electron donation.
Spatial precision of a catalytic carboxylate of F1‐ATPase β subunit probed by introducing different carboxylate‐containing side chains
Authors: Amano Toyoki , Tozawa Kaeko , Yoshida Masasuke , Murakami Hideto
Published: July 2002

Combining mutation and chemical modification, we have introduced Asp, Gln, Cys, S‐carboxymethylcysteine (Cax) and S‐carbamoylmethylcysteine (Cam) into the positions of Glu190 and Glu201 of the β subunit of F1‐ATPase from the thermophilic Bacillus PS3. The steady‐state ATPase activities of α3β3γ complexes containing these changed β subunits were 12% (E190Cax), 7% (E190D), 3% (E190Cam), <1% (E190C), <1% (E190Q), and 73% (E201D), 40% (E201Cax), 25% (E201C), 20% (E201Q), 4% (E201Cam), of that of the wild‐type α3β3γ complex. For the complexes containing E190C or E190Q, even the ability of single‐site catalysis was lost. Thus, the presence of a carboxylate at 190 (but not at 201) is absolutely required for catalysis and its spatial precision is very strict. Analysis of inactivation of the complexes by dicyclohexylcarbodiimide suggests that Glu190 and Glu201 are interacting in the F1‐ATPase.
Activation of the mouse cytokeratin A (endo A) gene in teratocarcinoma F9 cells by the histone deacetylase inhibitor Trichostatin A
Authors: Miyashita Tomoyuki , Yamamoto Hideyuki , Nishimune Yoshitake , Nozaki Masami , Morita Takashi , Matsushiro Aizo
Published: July 2002

Treatment of cultured cells with sodium butyrate, that is the histone deacetylase inhibitor, induces the histone hyperacetylation and the expressions of various mammalian genes without affecting the level of protein synthesis. However, butyrate is a non‐specific inhibitor of deacetylase because of its effects on various other enzymes and nuclear proteins other than histones. On the other hand, Trichostatin A (TSA) was recently found to be a potent and specific inhibitor of histone deacetylase. We examined the effect of TSA on the expression of mouse cytokeratin A (endo A). TSA increased endo A expression in F9 cells, and was efffective at a much lower concentration than sodium butyrate. We also examined the changes of chromatin structure induced by the two drugs by a DNase I‐hypersensitivity assay. Both drugs induced the formation of a DNase I‐hypersensitive site (DH site) in only the promoter region. The precise mechanism(s) by which the two drugs increase endo A gene expression is unknown, but these results suggest that endo A expression is induced by inhibition of histone deacetylase and that the effect is at the transcriptional level.
Enhancing effect of the 3′‐untranslated region of tobacco mosaic virus RNA on protein synthesis in vitro
Authors: Zeyenko Vladimir V. , Ryabova Lyubov A. , Gallie Daniel R. , Spirin Alexander S.
Published: July 2002

In order to test the enhancing effect of the 3′‐terminal untranslated region (3′‐UTR) of tobacco mosaic virus (TMV) RNA on protein synthesis in vitro we used a chimeric mRNA construct containing TMV 5′‐UTR (Ω) and firefly luciferase mRNA. The addition of the TMV 3′‐UTR to the chimeric mRNA construct results in a more than 3‐fold stimulation of the synthesis of the functionally active protein in the wheat germ cell‐free translation system. We have demonstrated that the proper length of the TMV 3′‐terminal part is important for efficient translation; elongation of the TMV tail by 160 vector‐derived nucleotides fully abolishes the stimulation effect of the TMV 3′‐UTR in vitro.
Polymannosylation to asparagine‐19 in hen egg white lysozyme in yeast
Authors: Kato Akio , Takasaki Hajime , Ban Masanori
Published: July 2002

Complementary DNA encoding hen egg white lysozyme (HEWL) was subjected to site‐directed mutagenesis to have the N‐glycosylation signal sequence (Asn19‐Tyr20‐Thr21) by substituting Arg with Thr at position 21. The mutant lysozyme (R21T) was expressed in Saccharomyces cerevisiae carrying the yeast expression plasmid inserting the mutant HEWL cDNA. The mutant lysozyme was expressed in the glycosylated forms which are mainly a polymannosyl form with a small amount of oligomannosyl form. The polymannosyl lysozyme was susceptible to Endo H cleavage of the carbohydrate chain. The length of the polymannose chain was predicted to be approximately 340 residues/mol of lysozyme from carbohydrate analysis. According to the estimation with low‐angle laser light scattering combined with HPLC, the average molecular weight of polymannosyl lysozyme was 75 kDa, which is consistent with the value obtained from the carbohydrate analysis. The size of polymannosyl lysozyme R21T is similar or somewhat larger than that of G49N reported previously. Thus, it was confirmed that the unusually large polymannose chain was attached to heterologous mutant lysozyme, regardless of the N‐linked position, in yeast.
Biochemical properties of attachment region binding protein ARBP
Authors: von Kries Jens P. , Rosorius Olaf , Buhrmester Hartmut , Strätling Wolf H.
Published: July 2002

ARBP (attachment region binding protein) is an abundant nuclear protein that specifically binds to matrix/scaffold attachment regions (MARs/SARs). Here we show by gel filtration and gradient sedimentation that ARBP has an elongated shape. The sedimentation coefficient was determined as only 2.1 S. Furthermore, limited proteolysis of ARBP in situ (in isolated nuclei) with several proteases generated limiting resistant peptides from 14.5 to 18 kDa, that retained the ability to bind MARs specifically. This indicates that these peptides encompass the DNA binding domain of ARBP.
Down‐regulation of non‐L‐, non‐N‐type (Q‐like) Ca2+ channels by Lambert‐Eaton myasthenic syndrome (LEMS) antibodies in rat insulinoma RINm5F cells
Authors: Magnelli V. , Grassi C. , Parlatore E. , Sher E. , Carbone E.
Published: July 2002

The action exerted on non‐L‐, non‐N‐type (Q‐like) Ca2+ channels by immunoglobulins G (IgGs) obtained from two patients with Lambert‐Eaton myasthenic syndrome (LEMS) was investigated in the rat insulinoma RINm5F cell line. LEMS IgGs reduced by 30–36% the whole‐cell Ba2+ currents through Q‐like Ca2+ channels at +10 mV without significantly modifying their voltage dependence and activation kinetics. Single‐ and multiple‐channel recordings in cell‐attached and outside‐out patches of cells treated with LEMS IgGs showed no significant changes of the channel elementary properties but rather a decreased number of active channels per patch. This suggests that Q‐like current depression by LEMS autoantibodies is mostly due to a down‐regulation of functioning Ca 2+ channels. In agreement with previous observations, LEMS IgGs also reduced by 20–33% the dihydropyridine‐sensitive (L‐type) Bat+ current. The suggested down‐regulation of Q‐like channels by LEMS IgGs in RINm5F cells may have a functional correlation with the depressive action of LEMS autoantibodies on the PIQ‐type Ca2+ channels controlling acetylcholine release from mammalian neuromuscular junctions.
Stabilization of the chlorophyll binding apoproteins, P700, CP47, CP43, D2, and D1, by synthesis of Zn‐pheophytin a in intact etioplasts from barley
Authors: Eichacker Lutz Andreas , Müller Bernd , Helfrich Michael
Published: July 2002

Chlorophyll a was compared with Zn‐pheophytin a for stabilization of chlorophyll binding apoproteins, P700, CP47, CP43, D2, and D1, in intact etioplasts from barley (Hordeum vulgare L.). Intact etioplasts were shown to effectively translate the chlorophyll apoproteins, to take up and esterify the exogenously added substrates, chlorophyllide a and Zn‐pheophorbide a, with geranylgeraniolpyrophosphate. For stabilization of P700, CP47, D2, and D1, the product, Zn‐pheophytin a, was shown to substitute for chlorophyll a. Stabilization of CP43 was selectively increased in the presence of Zn‐pheophytin a. The degree of stabilization was shown to depend on the amount of newly synthesized Zn‐pheophytin a and on the central atom of the chlorophyll molecule.
Evidence for a multi‐domain structure for hormone‐sensitive lipase
Authors: Smith Gabriele M. , Garton Andrew J. , Aitken Alastair , Yeaman Stephen J.
Published: July 2002

Hormone‐sensitive lipase (HSL) is a multi‐functional enzyme involved in several aspects of lipid metabolism. Limited tryptic digestion of HSL led to selective loss of activity against lipid substrates but not against the water‐soluble substrate, p‐nitrophenyl butyrate. Following labelling of the active site of HSL with either [3H]di‐isopropylfluorophosphate or [14C]orlistat, tryptic digestion of HSL generated a stable radiolabelled domain of molecular mass approx. 17.6 kDa, consistent with this representing a catalytic domain of HSL capable of hydrolysing water‐soluble but not lipid substrates. Following phosphorylation of HSL by cyclic AMP‐dependent protein kinase, limited tryptic digestion produced a stable phosphorylated domain of molecular mass 11.5 kDa. Based on these experimental data a model for a domain structure of HSL is proposed.
Kinesin subfamily UNC104 contains a FHA domain: boundaries and physicochemical characterization
Authors: Westerholm-Parvinen Ann , Vernos Isabelle , Serrano Luis
Published: July 2002

By sequence analysis we show that the U104 domain found in the UNC104 subfamily of kinesins is a forkhead homology‐associated domain (FHA). A combination of limited proteolysis, mass spectroscopy, and physicochemical analysis define this domain as a genuine autonomously folding domain. Our data show that the FHA domain is shorter than previously reported since the C‐terminal α‐helix is not part of its minimum core. Key amino acids postulated to recognize phosphorylated residues are conserved. These data suggest that the kinesin FHA domains are functional domains involved in protein–protein interactions regulated by phosphorylation.
Opposing regulation of B cell receptor‐induced activation of mitogen‐activated protein kinases by CD45
Authors: Ogimoto Mami , Arimura Yutaka , Katagiri Tatsuo , Mitomo Katsuyuki , Woodgett James R. , Nebreda Angel R. , Mizuno Kazuya , Yakura Hidetaka
Published: July 2002

In this study, we examined the contribution made by CD45 to B cell antigen receptor (BCR)‐induced activation of mitogen‐activated protein kinase (MAPK) family members. We found that CD45 negatively regulated BCR‐induced c‐Jun NH2‐terminal kinase (JNK) and p38 activation in immature WEHI‐231 cells, whereas in mature BAL‐17 cells, CD45 positively regulated JNK and p38 activation and negatively regulated extracellular signal‐regulated kinase activity. Furthermore, cooperative action of JNK and p38 dictated BCR‐induced inhibition of growth. Thus, CD45 appears to differentially regulate BCR‐induced activation of MAPK members, and can exert opposing effects on JNK and p38 in different cellular milieu, controlling the B cell fate.
Guide DNA technique reveals that the protein component of bacterial ribonuclease P is a modifier for substrate recognition
Authors: Tanaka Terumichi , Baba Hideo , Hori Yoshiaki , Kikuchi Yo
Published: July 2002

We developed a guide DNA technique with which the cleavage efficiency of pre‐tRNA substrate raised in the RNase P reaction. The 20‐mer guide DNAs hybridizing to the upstream region of the cleaving site enhanced the cleavage reactions of RNA substrates by Escherichia coli RNase P. This guide DNA technique was also applicable to cleavage site selection by choosing the DNA‐hybridizing site. Results showed that RNase P accepts DNA/RNA double‐stranded 5′‐leader region with high catalytic efficiency as well as single‐stranded RNA region in pre‐tRNAs as substrates, which suggests that the protein component of bacterial RNase P prefers bulky nucleotides. The protein component did not affect the normal 5′‐processing reaction of pre‐tRNAs, but enhanced the mis‐cleaving (hyperprocessing) reactions of tRNA in non‐cloverleaf folding. Our results suggested that the protein component of RNase P is a modifier for substrate recognition.
Functional evidence for the identification of an Arabidopsis clathrin light chain polypeptide
Authors: Scheele Urte , Holstein Susanne E.H
Published: July 2002

Clathrin light chains (CLCs) are regulatory subunits of clathrin triskelia. Based on homology searches in Arabidopsis thaliana data bases we have identified three putative CLC clones, and have focused on the one with the highest homology to mammalian CLC sequences. Analysis of its sequence has revealed coiled‐coil structures within a region that corresponds to the clathrin heavy chain‐binding site. In addition there is a stretch of acidic amino acids, which is required for the regulatory function of CLC in clathrin assembly. This putative plant CLC ortholog, expressed in bacteria as a glutathione‐S‐transferase‐ and myc‐tagged fusion protein, was shown to bind to CLC‐free recombinantly expressed mammalian clathrin hubs. In contrast, purified native mammalian triskelia with endogeneous CLC did not bind the recombinant putative plant CLC. Based on the conserved sequences between the three Arabidopsis candidates it appears that plants, unlike mammals, may have more than two CLCs.
Molecular heterogeneity of the cDNA encoding a 74‐kDa regulatory subunit (B″ or δ) of human protein phosphatase 2A1
Authors: Tanabe Osamu , Gomez Gloria A , Nishito Yasumasa , Usui Hirofumi , Takeda Masao
Published: July 2002

Two cDNAs for possible splicing variants of a 74‐kDa regulatory subunit (B″ or δ) of human protein phosphatase 2A, were isolated. These variants were identified from human cerebral cortex by library screening and PCR, and designated δ1 and δ3 isoforms, while the previously reported isoform [Tanabe et al. (1996) FEBS Lett. 379, 107–111] was designated δ2. Compared with the δ2 isoform, the δ1 isoform contained a 32‐residue insertion beginning at residue 84, and consisted of 602 amino acids in all. The δ3 isoform lacked a 74‐residue sequence corresponding to residues 1083 of the δ2 isoform, and consisted of 496 amino acids. Using isoform‐specific antipeptide antisera, the 74‐kDa subunit (B″ or δ) originally purified from human erythrocytes was identified as the δ1 isoform.
Antioxidant activity of allopurinol on copper‐catalysed human lipoprotein oxidation
Authors: Lapenna Domenico , de Gioia Sergio , Ciofani Giuliano , Cuccurullo Franco
Published: July 2002

We found that allopurinol, at therapeutically relevant concentrations (9–58 μM), significantly counteracted copper‐catalysed human non‐HDL lipoprotein oxidation, as assessed by thiobarbituric acid reactant content and kinetics of conjugated diene formation. Oxypurinol was ineffectual. Both drugs had no activity on metal‐independent, peroxyl radical‐induced lipoprotein oxidation. Specific fluorescence‐quenching experiments revealed that only allopurinol could interact with copper antagonizing metal binding to lipoproteins. Thus, therapeutic allopurinol concentrations can inhibit copper‐catalysed lipoprotein oxidation through metal complexation, suggesting some antioxidant‐antiatherogenic activity of the drug in vivo.
Evidence for a crosslink between c‐heme and a lysine residue in cytochrome P460 of Nitrosomonas europaea
Authors: Arciero David M. , Hooper Alan B.
Published: July 2002

Sequence analysis of a purified heme‐containing tryptic chromopeptide from cytochrome P460 revealed two predominant amino acid residues per cycle. Two peptides present in the chromopeptide with the sequences NLPTAEXAAXHK and DGTVTVXELVSV. Comparison of the data to the gene sequence for the protein revealed that the gaps in the first peptide (indicated by X's) code for C residues, confirming the prediction of a c‐heme binding motif. The gap in the sequence in the second peptide at cycle 7 is predicted by the gene sequence to be a K. The results suggest that the lysine residue is crosslinked in some manner to the porphyrin macrocycle, possibly mimicking the tyrosine crosslink found for the heme P460 of HAO. While a common role for the crosslinked residues in HAO and cytochrome P460 is difficult to ascertain due to the dissimilarities in side chain structure, it may be related to the similar pK
a values for lysine and tyrosine.
Functional analysis of a rat sodium channel carrying a mutation for insect knock‐down resistance (kdr) to pyrethroids
Authors: Vais H , Williamson M.S , Hick C.A , Eldursi N , Devonshire A.L , Usherwood P.N.R
Published: July 2002

Pyrethroid insensitivity in resistant (kdr) insects has been correlated with a leucine to phenylalanine replacement in the S6 transmembrane segment of domain II of the axonal sodium channel α(para)‐subunit. An α‐subunit of rat brain type II sodium channel containing this mutation has been expressed and its sensitivity to permethrin compared with that of the wild‐type channel. The steady‐state activation curve of the mutant was shifted 14 mV in the depolarizing direction. We propose that an equivalent shift of the sodium current activation curve in kdr insects could account for their low sensitivity to permethrin toxicity.
Thermodynamic characterizations of an intramolecularly hydrogen bonded C5‐structure across proteinogenic residue
Authors: Ashish  , Kishore R
Published: July 2002

Thermodynamic investigations of a smallest possible intramolecularly hydrogen bonded C5‐structure, across a Thr residue, in model peptides Boc‐Xxx‐Thr‐NH2 (Xxx=Ile, 1 or Leu, 2), indicated unusual thermal stability of the structure in non‐polar medium. An analysis of van't Hoff plots, constructed from variable temperature 1H NMR data, yielded the thermodynamic parameters of a hydrogen bonded five‐membered ring. The non‐significance of the spatial organizations of the preceding CδH3 bearing hydrophobic proteinogenic residue on the thermal stability of the C5‐structure has been observed. The results revealed that the contribution of this element of secondary structure is quantifiable and the stability appeared to be roughly comparable to other intramolecularly hydrogen bonded reverse turn structures frequently observed in polypeptides and proteins.
NSC1: a novel high‐current inward rectifier for cations in the plasma membrane of Saccharomyces cerevisiae
Authors: Bihler Hermann , Slayman Clifford L , Bertl Adam
Published: July 2002

The plasma membrane of the yeast Saccharomyces cerevisiae possesses a non‐specific cation ‘channel', tentatively dubbed NSC1, which is blocked by normal (mM) calcium and other divalent metal ions, is unblocked by reduction of extracellular free divalents below ∼10 μM, and is independent of the identified potassium channel and porters in yeast, Duk1p, Trk1p, and Trk2p. Ion currents through NSC1, observed by means of whole‐cell patch recording, have the following characteristics: Large amplitude, often exceeding 1 nA of K+/cell at −200 mV, in tetraploid yeast, sufficient to double the normal intracellular K+ concentration within 10 s; non‐saturation at large negative voltages; complicated activation kinetics, in which ∼50% of the total current arises nearly instantaneously with a voltage‐clamp step, while the remainder develops as two components, with time constants of ∼100 ms and ∼1.3 s; and voltage independence of both the activation time constants and the associated fractional current amplitudes.
Observing the deoxy myoglobin and hemogobin signals from rat myocardium in situ
Authors: Tran Tuan-Khanh , Kreutzer Ulrike , Jue Thomas
Published: July 2002

1H NMR proximal histidyl NδH signals of deoxy hemoglobin (Hb) and myoglobin (Mb) are distinguishable in the rat myocardium in situ. In the normoxic resting state, the blood and tissue pO2 is sufficient to saturate both Mb and Hb. No deoxy Mb or Hb signals are detected. Under 12% inspired O2, the erythrocyte Hb is partially desaturated and yields the α and β proximal histidyl NδH signals of deoxy Hb. The detection of the Hb signals clarifies the debate about the NMR visibility of erythrocyte Hb in vivo and augments the strategy to observe tissue pO2.
Marked differences between avian and mammalian testicular cells in the heat shock induction and polyadenylation of Hsp70 and ubiquitin transcripts
Authors: Mezquita Belen , Mezquita Cristóbal , Mezquita Jovita
Published: July 2002

Mammalian male germ cells undergo apoptosis at the body's internal temperature of 37°C. Birds, however, are unique among homeothermic animals in developing spermatogenesis at the elevated avian internal body temperature of 40–41°C. To shed light on the mechanisms that maintain an efficient avian spermatogenesis at elevated temperatures we compared, in mouse and chicken testicular cells, the expression of genes that are essential for stress resistance: Hsp70 and ubiquitin. While the expression of Hsp70 and ubiquitin did not change upon heat shock in mouse testicular cells, both the amount and polyadenylation of Hsp70 and ubiquitin transcripts increased when male germ cells from adult chicken testis were exposed to elevated temperatures.
Erythrosin 5′‐isothiocyanate labels Cys549 as part of the low‐affinity ATP binding site of Na+/K+‐ATPase1
Authors: Linnertz Holger , Kost Holger , Obsil Tomas , Kotyk Arnost , Amler Evzen , Schoner Wilhelm
Published: July 2002

The high‐affinity E1ATP site of Na+/K+‐ATPase labeled with fluorescein 5′‐isothiocyanate and its E2ATP site labeled with erythrosin 5′‐isothiocyanate (ErITC), as was shown recently [Linnertz et al. (1998) J. Biol. Chem. 273, 28813–28821], reside on separate and adjacent catalytic α subunits. This paper provides evidence that specific labeling of the E2ATP binding site with ErITC resulted in a modification of the Cys549 residue in the tryptic fragment with the sequence Val545‐Leu‐Gly‐Phe‐Cys549‐His550. Hence, Cys549 is part of or close to the low‐affinity E2ATP binding site of Na+/K+‐ATPase.
Carboxyatractyloside increases the effect of oleate on mitochondrial permeability transition
Authors: Chávez Edmundo , Zazueta Cecilia , Garcı́a Noemı́
Published: July 2002

Addition of a low concentration of carboxyatractyloside (0.075 μM) renders mitochondria susceptible to the opening of the non‐specific pore by 5 μM oleate, in a cyclosporin A‐sensitive fashion. Matrix Ca2+ efflux as well as collapse of the transmembrane potential reveal permeability transition. The effect of oleate is reached after the titration, by carboxyatractyloside, of 38 pmol of adenine nucleotide translocase per mg mitochondrial protein. We propose that permeability transition may result from an additive action of carboxyatractyloside plus oleate on the ADP/ATP carrier.
Phosphorylation of tau protein by recombinant GSK‐3β: pronounced phosphorylation at select Ser/Thr‐Pro motifs but no phosphorylation at Ser262 in the repeat domain
Authors: Godemann R. , Biernat J. , Mandelkow E. , Mandelkow E.-M.
Published: July 2002

Glycogen synthase kinase‐3β (GSK‐3β) has been described as a proline‐directed kinase which phosphorylates tau protein at several sites that are elevated in Alzheimer paired helical filaments. However, it has been claimed that GSK‐3β can also phosphorylate the non‐proline‐directed KXGS motifs in the presence of heparin, including Ser262 in the repeat domain of tau, which could induce the detachment of tau from microtubules. We have analyzed the activity of recombinant GSK‐3β and of GSK‐3β preparations purified from tissue, using two‐dimensional phosphopeptide mapping, immunoblotting with phosphorylation‐sensitive antibodies, and phosphopeptide sequencing. The most prominent phosphorylation sites on tau are Ser396 and Ser404 (PHF‐1 epitope), Ser46 and Thr50 in the first insert, followed by a less efficient phosphorylation of other Alzheimer phosphoepitopes (antibodies AT‐8, AT‐270, etc). We also show that the non‐proline‐directed activity at KXGS motifs is not due to GSK‐3β itself, but to kinase contaminations in common GSK‐3β preparations from tissues which are activated upon addition of heparin.