bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucose‐induced cAMP signaling in Saccharomyces cerevisiae is mediated by the CDC25 protein
Authors: Munder Thomas , Küntzel Hans
Published: July 2002

Functional mapping of the cell cycle START gene CDC25 has revealed two domains which are dispensable for viability (germination and growth in glucose media), but are essential for sporulation and differentially involved in glucose‐induced cAMP signaling. The transient rise of cAMP is completely prevented by various deletions within the amino‐terminal half (α domain) of the CDC25 gene product. In contrast, the deletion of the carboxy‐terminal 38 residues (β2 domain) results in a rapid, but persisting, rise of cAMP. Our data suggest that the α domain of the CDC25 protein is involved in glucose signal transduction, whereas the β2 domain is required for downregulating the cAMP control chain.
Long‐range intramolecular electron transfer in Rhus vernicifera stellacyanin: A pulse radiolysis study
Authors: Farver Ole , Pecht Israel
Published: July 2002

The (NH3)5Ru‐modified stellacyanin Ru(III)2‐St[Cu(II)], where two ruthenium ions are coordinated to imidazole residues of His‐32 and 100, has been synthesized. CO−
2 radicals, generated by pulse radiolysis, react with the modified protein yielding an intermediate Ru(II)‐St[Cu(II)]. The rate of molecular electron transfer within this complex Ru(II)‐St[Cu(II)]
k
Ru(III)‐St[Cu(I)] has been determined, k
u = 0.05 s−1 at 18°C, pH 7.0. The Ru(His‐32/100) to Cu(II) separation distance is relatively large (∼ 18 Å), and the driving force is low (10 kJ·mol−1), hence the correspondingly slow rate. This result is compared with those obtained in earlier studies of intramolecular fixed‐distance electron transfer reactions in blue copper proteins.
Transducin GTPase provides for rapid quenching of the cGMP cascade in rod outer segments
Authors: Arshavsky V.Yu. , Antoch M.P. , Lukjanov K.A. , Philippov P.P.
Published: July 2002

The role of transducin GTPase in rapid cGMP phosphodiesterase quenching was studied by simultaneous registration of GTP hydrolysis and phosphodiesterase activity in the same rod outer segments (ROS) preparation. The results thus obtained allow the conclusion that: (i) phosphodiesterase quenching coincides with transducin‐bound GTP hydrolysis independently of ROS concentration; (ii) an increase in the ROS concentration results in the acceleration of cascade quenching due to the existence of a GTPase accelerating mechanism in ROS; (iii) approximation to physiological conditions (protein concentration, temperature) provides a transducin GTPase rate equal to 1–2 turnovers per second i.e., sufficiently high for satisfying the real rate of photoresponse reversion in dark‐adapted rods.
Binding of plasminogen to Escherichia coli adhesion proteins
Authors: Parkkinen Jaakko , Korhonen Timo K.
Published: July 2002

Immobilization of plasminogen via its lysine‐binding sites is regarded as a prerequisite for its activation and function in fibrinolysis and pericellular proteolysis. In the present study, the interaction of plasminogen with fimbriae found on Escherichia coli strains causing invasive human infections was studied. Plasminogen displayed concentration‐dependent and saturable binding to immobilized type 1 fimbriae and, several fold lower binding to P and S fimbriae. The binding to fimbriae was effectively inhibited by ε‐aminocaproic acid indicating that it was mediated by the lysine‐binding sites of plasminogen. Binding studies with mutated fimbriae and inhibition tests indicated that the interaction was not dependent on the lectin subunit of the fimbriae. These results indicate the existence of a novel type of host‐microbe interaction which may be important in the invasion by bacteria of host tissues.
Towards the development of antimicrobial drugs acting by inhibition of pathogen attachment to host cells: A need for polyvalency
Authors: Matrosovich Mikhail N.
Published: July 2002

The development of inhibitors of microbial attachment to target cells has been proposed recently as a possible novel approach to antimicrobial chemoprophylaxis and treatment. In this paper an attempt is made to contend that such artificial inhibitors must be polyvalent, i.e. capable of binding to the pathogen or its target by multiple bonds.
Two early genes of bacteriophage T5 encode proteins containing an NTP‐binding sequence motif and probably involved in DNA replication, recombination and repair
Authors: Blinov V.M. , Koonin E.V. , Gorbalenya A.E. , Kaliman A.V. , Kryukov V.M.
Published: July 2002

It is demonstrated, by computer‐assisted analysis, that T5 bacteriophage early genes D10 and D13 encode proteins containing the purine NTP‐binding sequence motif. The D10 gene product is shown to be a member of a recently characterized superfamily of (putative) DNA and RNA helicases. The D13 gene product is related, at a statistically significant level, to the gene 46 product of bacteriophage T4 which is a component of an exonuclease involved in phage DNA replication, recombination and repair. A lower but also significant degree of sequence similarity was detected between the gene D12 product of T5 and the gene 47 product of T4, the second component of the same nuclease. It is hypothesized that both D10 and D13 gene products of T5 might be NTPases, possibly DNA‐dependent, mediating NTP‐consuming steps during phage DNA replication, recombination and/or repair.
Determination of PQQ in quinoproteins with covalently bound cofactor and in PQQ‐derivatives
Authors: van der Meer Robert A. , Mulder Antonia C. , Jongejan Jacob A. , Duine Johannis A.
Published: July 2002

Application of the so‐called hexanol extraction procedure for PQQ determination, originally based on detachment of the cofactor from quinoproteins and conversion into PQQ‐5,5‐dihexyl ketal, leads in several cases to a number of products due to uncontrollable esterification. The present modified procedure, detaching the covalently bound cofactor and converting it into 4‐hydroxy‐5‐hexoxy‐pyrroloquinoline, was tested on a number of proteins. Only the expected product was obtained for the known quinoproteins, in a quantitative yield, as revealed by comparison with the values determined with the hydrazine method. Thus this independent method confirmed that bovine serum amine oxidase, porcine kidney diamine oxidase, dopamine β‐hydroxylase from bovine adrenal medulla, methylamine dehydrogenase from Thiobacillus
inversutus, glutamate decarboxylase from Escherichia coli, and 3,4‐dihydroxyphenylalanine decarboxylase from pig kidney are really quinoproteins. Quantitative conversion was also achieved for condensation and addition products of PQQ (PQQ‐acetone, PQQH2, PQQ‐oxazole, PQQ‐dinitrophenylhydrazone, and PQQ‐tryptophan). In view of this conversion and the fact that catalytic activity of PQQ is not required, the method seems suited to investigate the distribution of the cofactor in eukaryotes, especially in mammals where it is almost certain that PQQ occurs only in derivatized form. Finally, just like the hydrazine method, the hexanol extraction procedure seems unable to keep the structure of the cofactor as it exists in the active site, intact, as demonstrated for the pro‐PQQ cofactor of methylamine dehydrogenase.
Cleavage of a synthetic COOH‐terminal oligopeptide of D1 precursor protein by a purified processing enzyme
Authors: Fujita S. , Inagaki N. , Ono T. , Inoue Y. , Satoh K.
Published: July 2002

A synthetic COOH‐terminal oligopeptide of D1 protein deduced from the spinach psbA gene (Asn‐325‐Gly‐353) was subjected to proteolytic digestion by purified processing enzyme of D1 protein [(1989) FEBS Lett. 246, 218–222] and the following two fragments were obtained as cleavage products: a COOH‐terminal 9‐amino‐acid fragment (Ala‐345‐Gly‐353) and an NH2‐terminal 10‐amino‐acid fragment (Asn‐325‐Arg‐334). It was concluded that: (i) the oligopeptide consisting of the COOH‐terminal 29‐amino‐acid sequence deduced from the spinach psbA gene provides the recognition domain for the processing enzyme; (ii) the cleavage takes place at the predicted processing site of native DI precursor protein (COOH side of Ala‐344); and (iii) another cleavage takes place at an additional site (COOH side of Arg‐334) for the synthetic substrate, but not for the native D1 precursor protein.
Acidic and basic fibroblast growth factors similarly regulate the rate of biosynthesis of rat astroblast proteins
Authors: Loret Camille , Laeng Pascal , Sensenbrenner Monique , Labourdette Gérard
Published: July 2002

Quiescent rat astroblasts in culture have been treated for various periods of time with acidic and basic fibroblast growth factors. Both factors elicited similar effects on the cell proliferation and glutamine synthetase activity. The rate of biosynthesis of the proteins analyzed on autoradiograms of polyacrylamide gels after two‐dimensional electrophoresis was also similarly modulated by the two growth factors. These results suggest that the two fibroblast growth factors act through the same membrane receptors on rat astroblasts in culture.
Amino acid sequence of CAP37, a human neutrophil granule‐derived antibacterial and monocyte‐specific chemotactic glycoprotein structurally similar to neutrophil elastase
Authors: Pohl Jan , Pereira H.Anne , Martin Nancy M. , Spitznagel John K.
Published: July 2002

We report the amino acid sequence of CAP37, a human neutrophil granule protein with antibacterial and monocyte‐specific chemotactic activity. CAP37 is a single‐chain protein consisting of 222 amino acid residues. It has three N‐glycosylation sites, at Asn residues 100, 114 and 145. Some species of CAP37 are glycosylated at all three sites; some at Asn‐114 alone, others at Asn‐114 and Asn‐110 or Asn‐145. CAP37 has 45% sequence identity to human neutrophil elastase, and 30–37% identity to several other granule serine proteinases. Despite these similarities, CAP37 is not a serine proteinase because the active site residues serine and histidine are replaced.
Structure‐function analysis of epidermal growth factor: site directed mutagenesis and nuclear magnetic resonance
Authors: Dudgeon T.J. , Cooke R.M. , Baron M. , Campbell I.D. , Edwards R.M. , Fallon A.
Published: July 2002

The role of leucine‐47 in determining the structure and activity of human epidermal growth factor was examined using site‐directed mutagenesis. Wild type protein and four variants in which Leu47 was replaced by valine, glutamate, aspartate and alanine were produced from yeast. 1H NMR experiments demonstrated that substitution of Leu47 had little effect on the protein structure. The observed reduction in receptor binding affinity caused by the substitutions could thus be attributed to perturbation of a residue directly involved in receptor interactions.
Interleukin‐1β inhibits glucokinase activity in clonal HIT‐T15 β‐cells
Authors: Beggs Mark , Beresford Guy , Clarke Jennifer , Mertz Robert , Espinal Joseph , Hammonds Peter
Published: July 2002

Interleukin‐1β (IL‐1β) has been implicated in the pathogenesis of insulin‐dependent diabetes mellitus. In the present study we have investigated the effects of IL‐1β on glucose metabolism in clonal HIT‐T15 β cells. In the short‐term (1 h), 25 IL‐1β significantly increased the rates of insulin release and glucose utilisation, but not glucose oxidation. In contrast, after 48 h, IL‐1β inhibited insulin release and glucose utilisation and oxidation. By assaying enzymes (hexokinase, glucokinase, pyruvate dehydrogenase, glucose 6‐phosphatase) and nucleotides (ATP, ADP) associated with the regulation of glycolysis and glucose oxidation, we conclude that the inhibitory effects of IL‐1β may be due to impaired glucokinase activity.
Exacerbation of rat adjuvant arthritis by intradermal injection of purified mammalian 14‐kDa group II phospholipase A2
Authors: Murakami Makoto , Kudo Ichiro , Nakamura Hideo , Yokoyama Yuichi , Mori Hiroshi , Inoue Keizo
Published: July 2002

Appreciable phospholipase A2 activity was detected in a hind paw homogenate from rats with adjuvant arthritis or carrageenan‐induced edema. The activity was neutralized by treatment with antibody raised against rat platelet secretory phospholipase A2, indicating that 14‐kDa group II phospholipase A2, was induced in the hind paw during the process of inflammation. Injection of purified rat platelet phospholipase A2 into the hind paw of rats with adjuvant‐induced arthritis resulted in exacerbation of edema in a dose‐dependent manner, whereas no effect was observed on either normal rats or animals with carrageenan‐induced edema under the same experimental conditions. These results suggest the importance of mammalian group II phospholipase A2, in the pathogenesis of some types of inflammation.
Eukaryotic DNA helicases
Authors: Thömmes Pia , Hübscher Ulrich
Published: July 2002

DNA is very stable in its double‐stranded form. For many processes of DNA metabolism, such as replication, repair, recombination and transcription, the DNA has to be brought transiently into a single‐stranded form. DNA helicases are enzymes capable of melting the hydrogen bonds of base pairs by using the energy of nucleoside‐5'‐triphosphate hydrolysis. This minireview focusses on the current knowledge of DNA helicases from eukaryotic cells.
Diverse actions of cadmium on the smooth muscle myosin phosphorylation system
Authors: Kostrzewska Anna , Sobieszek Apolinary
Published: July 2002

The effects of cadmium (Cd) on smooth muscle myosin phosphorylation have been investigated using an in vitro system comprising myosin filaments containing endogenous calmodulin (CM) and myosin light chain kinase (MLCKase). In the absence of calcium (Ca), Cd as well as some other divalent cations caused no activation of phosphorylation. However, when at least one (or possibly two) Ca2+ were bound per CM, the addition of 10 μM to 40 μM Cd2+ resulted in a 2 to 3 fold acceleration of the phosphorylation rate. Higher Cd concentrations caused inhibition of the system independent of Ca2+ concentration through the formation of Cd‐ATP complexes. These results explain some previously controversial data on the complex effects of Cd in intact smooth muscles.
Examination of elongation factor Tu for aluminum fluoride binding sites using fluorescence and 19F‐NMR methodologies
Authors: Hazlett Theodore L. , Higashijima Tsutomu , Jameson David M.
Published: July 2002

This article reports on a comparison or the interaction of AP3+ and F− with two GTP‐binding proteins, elongation factor Tu (EF‐Tu) and the hormone sensitive regulatory protein (G protein) Goα. The methodologies chosen to elucidate possible between protein and aluminum fluoride were fluorescence spectroscopy and nuclear magnetic resonance (19F‐NMR). Both proteins have tryptophan residues near their nucleotide binding sites, the purported site of aluminum fluoride interaction. It has been assumed for G proteins (including Goα) that aluminum fluoride, in the presence of Mg2+, mimics the magnesium coordinated γ‐phosphate group for the GDP‐form of the protein and shifts the protein's conformation toward the active GTP‐form. Indeed, changes in intrinsic fluorescence of Goα affected by aluminum fluoride are observed. The presence of aluminum fluoride did not affect the intrinsic fluorescence, spectra of lifetimes, of EF‐Tu·GDP. 19F‐NMR was then used to directly test for bound F−. Fluoride alone or in the presence of either protein gave a single 19F‐NMR peak at −10 ppm, characteristic of free F−. With the addition of aluminum to the protein and F− samples a second peak, shifted upfield from the first to −29 ppm, was observed for Goα·GDP. This second peak, which has been assigned to protein‐bound F−, was not observed for EF‐Tu·GDP. These observations show that the interaction of Al3+ and F−, in the presence of Mg2+, may be quite different between the hormone‐sensitive G proteins, which bind aluminum fluoride, and the GTP‐binding proteins as a whole, which include EF‐Tu. Care must therefore be exercised when structural data on the elongation factor, specifically on the nucleotide site, are used to interpret data or compose models intended to describe the hormone‐sensitive regulatory G proteins.
Biogenesis of secretory granules Implications arising from the immature secretory granule in the regulated pathway of secretion
Authors: Tooze Sharon A.
Published: July 2002

In endocrine cells the regulated secretion of hormones, peptides, enzymes and neurotransmitters into the external medium occurs when mature secretory granules fuse with the plasma membrane. Secretory granules form at the trans‐Golgi network (TGN) by envelopment of the dense‐core aggregate of regulated secretory proteins by a specific membrane. The secretory granules initially formed at the TGN, referred to here as immature secretory granules, are morphologically and biochemically distinct from mature secretory granules. The functional similarities and differences between the immature secretory granule and the mature secretory granule, and the events involved in the maturation of the secretory granules are briefly discussed.
Supramolecular structure of the recombinant murine small heat shock protein hsp25
Authors: Behlke Joachim , Lutsch Gudrun , Gaestel Mathias , Bielka Heinz
Published: July 2002

The size and shape of the recombinant murine small heat shock protein, hsp25, have been analyzed by hydrodynamic and electron microscopic methods. According to these studies recombinant hsp25 exists in large complexes with a sphere‐like shape and diameters of 15–18 nm. The molecular mass of these complexes amounts to about 730 kDa indicating that they are composed of about 32 monomers.
Differential glycosylation of N‐POMC1–77 regulates the production of γ3‐MSH by purified pro‐opiomelanocortin converting enzyme A possible mechanism for tissue‐specific processing
Authors: Birch Nigel P. , Estivariz Fernando E. , Bennett Hugh P.J. , Lob Y.Peng
Published: July 2002

The amino terminus of bovine pro‐opiomelanocortin (N‐POMC1–77) is partially processed in the intermediate lobe of the pituitary to N‐POMC1–49 and lys‐γ3 ‐melanotropin. Two pools of N‐POMC1–77 were isolated which were differentially glycosylated at threonine45, while N‐POMC1–49 isolated from bovine intermediate lobe extracts existed in a non‐glycosylated form. This suggested that differential O‐linked glycosylation of N‐POMC1–77 may regulate cleavage at the Arg49‐Lys50 processing site. We tested this hypothesis by incubating N‐POMC1–77 glycoforms with purified pro‐opiomelanocortin converting enzyme. Only non‐O‐glycosylated N‐POMC1–77 and O‐glycosylated N‐POMC1–77 with truncated oligosaccharide sidechains were sensitive to cleavage and generated predominantly lys‐γ3 ‐melanotropin, identified by high‐performance liquid chromatography. These data provide the first functional evidence to support a role for differential O‐linked glycosylation in the regulation of the processing of the N‐terminus of bovine POMC.
Characterization of a prolyl endopeptidase from spinach thylakoids
Authors: Kuwabara Tomohiko
Published: July 2002

A prolyl endopeptidase (PEPase, EC 3.4.21.26) that specifically cleaves the 18‐kDa protein of photosystem II was extracted from photosystem II membranes with 1 M NaCl. Proteolytic activity measured with artificial substrates was less than a quarter of that with the protein. Studies on inhibition of the proteolysis by an artificial substrate suggested that the protease recognizes the scissile prolyl bond. The protease was inhibited by CuCl2, but not by diisopropyl fluorophosphate or p‐chloromercuriphenylsulfonic acid. These findings suggest that the protease represents a new class of PEPase. The specificity of the enzyme is discussed in relation to the structure of the 18‐kDa protein.
Retroviral‐mediated transfer of a dystrophin minigene into mdx mouse myoblasts in vitro
Authors: Dunckley M.G. , Love D.R. , Davies K.E. , Walsh F.S. , Morris G.E. , Dickson G.
Published: July 2002

We have demonstrated expression of a 6.3 kb Becker muscular dystrophy (BMD) human dystrophin cDNA following retroviral‐mediated transduction of cultured myoblasts from the dystrophin‐deficient mdx mouse. The truncated dystrophin protein was localised to the sarcolemma of differentiated myotubes by antibodies against the C‐terminus of the molecule, and produced an identical immunostaining pattern to that observed in control myotubes expressing normal endogenous dystrophin. These results indicate that retroviral‐mediated gene transfer may be useful for experimental in vivo studies on the complementation of dystrophin gene mutations.
Protein deamidase from germinating wheat grains
Authors: Vaintraub Iosif A. , Kotova Ludmila V. , Shaha Ranajid
Published: July 2002

A new enzyme catalyzing the deamidation of seed storage proteins was found in germinating wheat grains and was partially purified. It also acts on egg lysozyme, horse hemoglobin and reduced RNAse, glutamine and Gly‐l‐Gin‐l‐Tyr. No activity was observed when using ovalbumin, serum albumin, RNAse, insulin, asparagine and an asparagine‐containing peptide. Only glutaminyl residues appear to be deamidated by this enzyme. It differs from transglutaminase and proved to be a true protein deamidase.
HSP70‐related 65 kDa protein of beet yellows closterovirus is a microtubule‐binding protein
Authors: Karasev Alexander V. , Kashina Anna S. , Gelfand Vladimir I. , Dolja Valerian V.
Published: July 2002

Beet yellows virus (BYV) genome encodes a 65 kDa protein homologous to the HSP70 family of cellular heat‐shock proteins (Agranovsky, A.A., Boyko, V.P., Karasev, A.V., Koonin, E.V. and Dolja, V.V. (1991) J. Mol. Biol. 217, 603–610). The respective gene was cloned and expressed in vitro yielding a product of the expected size (p65). This product was found to bind to the purified microtubules with a binding constant of 4 × 10−7 M. The binding of p65 was stimulated if ATP presented in the translation mixture was hydrolyzed by apyrase. Removal of the short C‐terminal domains of α‐ and β‐tubulin by subtilisin digestion abolished the binding, demonstrating its specificity. The possible role of p65 association with microtubules in the movement of virus within and/or between plant cells is proposed.
CDC25‐dependent induction of inositol 1,4,5‐trisphosphate and diacylglycerol in Saccharomyces cerevisiae by nitrogen
Authors: Schomerus Christof , Küntzel Hans
Published: July 2002

The addition of ammonium sulfate to starved yeast cells leads to a 3‐ to 4‐fold rapid increase of the second messengers inositol 1,4,5‐trisphosphate (IP3) and diacylglycerol (DAG), the products of phosphoinositide‐specific phospholipase C (PI‐PLC). This response is reduced by dissecting the RAS‐activating Cdc25 protein, and is completely abolished by the cdc25‐1 mutation even at permissive temperature. Starved cdc25‐1 mutant cells have a strongly reduced IP3 content, but an at least 10‐fold increased DAG level compared to the isogenic wild‐type strain. NH4 does not stimulate cAMP synthesis, and glucose does not induce IP3 and DAG. Our data suggest that the Cdc25 protein controls a nitrogen‐specific signalling pathway involving the effector PI‐PLC, in addition to the glucose‐induced activation of adenylyl cyclase (AC).
Construction and functional analysis of hybrid interleukin‐6 variants Characterization of the role of the C‐terminus for species specificity
Authors: Leebeek Frank W.G. , Fowlkes Dana M.
Published: July 2002

We have constructed several hybrid human interleukin‐6 (IL‐6) variants in which the carboxyl‐terminus, which includes a receptor binding site of IL‐6 has been replaced with the C‐terminus of various proteins homologous to human IL‐6, IL‐6 hybrids with the C‐terminus of human growth hormone and human granulocyte‐colony stimulating factor maintain part of the biological activity of human IL‐6. Replacing the C‐terminus of human IL‐6 with the C‐terminus of mouse and rat IL‐6 resulted in a normal or increased activity on a mouse cell line; however, this gave a low (to 200‐fold less) activity on a human cell line compared to wild‐type human IL‐6. We therefore conclude that the C‐terminus of IL‐6 plays in important role in the species specificity of IL‐6.
Differential role of four cysteines on the activity of a low Mr phosphotyrosine protein phosphatase
Authors: Chiarugi Paola , Marzocchini Riccardo , Raugei Giovanni , Pazzagli Claudia , Berti Andrea , Camici Guido , Manao Gianpaolo , Cappugi Gianni , Ramponi Giampietro
Published: July 2002

In this paper we describe the construction or five mutants of a bovine liver low M
r phosphotyrosine protein phosphatase (PTPase) expressed as a fusion protein with the maltose binding protein in E. coli. Almost no changes in the kinetic parameters were observed in the fusion protein with respect to the native PTPase. Using oligonucleotide‐directed mutagenesis Cys‐17, Cys‐62 and Cys‐145 were converted to Ser while Cys‐12 was converted to both Ser and Ala. The kinetic properties of the mutants, using p‐nitrophenyl phosphate as substrate, were compared with those of the normal protein fused with the maltose binding protein or E. coli; both of the Cys‐12 mutants showed a complete loss of enzymatic activity while the specific activity of the Cys‐17 mutant was greatly decreased (200‐fold). The Cys‐62 mutant showed a 2.5‐fold decrease in specific activity, while the Cys‐145 mutant remained almost unchanged. These data confirm the involvement of Cys‐12 and Cys‐17 in the catalytic site and suggest that Cys‐62 and Cys‐145 mutations may destabilise the structure of the enzyme.
Characterization of NCAM diversity in cultured neurons
Authors: Gegelashvili Georgi , Andersson Anna-Maria , Schousboe Arne , Bock Elisabeth
Published: July 2002

A single transcript of the NCAM gene undergoes differential processing resulting in a multiplicity of mRNAs and their translation products. In this study, the diversity of NCAM in rat primary neuronal cultures was investigated utilizing immuno‐ and Northern blot analyses. NCAM polypeptides of 190 kDa (NCAM‐A) and 135 kDa (NCAM‐B) were shown to be associated with the neuronal phenotype. These data were confirmed by Northern blotting, which in both neocortical neurons and cerebellar granule neurons revealed mRNA classes of 7.4 kb and 6.7 kb encoding for NCAM‐A and ‐B, respectively. However, oligonucleotide probes, specific for selected exons or exon combinations, revealed special features of cerebellar granule neurons as compared to neocortical neurons: expression of 4.3 kb NCAM mRNA, a relatively low amount of VASE‐containing variants, and an apparent lack of mRNA species containing exons a and an AAG insert between exons 12 and 13. Distinct patterns of NCAM mRNA may putatively be related to the regional origin and functional specificity of the investigated neurons.
Identification of a putative active site residue in the exo‐β‐(1,3)‐glucanase of Candida albicans
Authors: Chambers Ross S. , Walden Adrian R. , Brooke Giles S. , Cutfield John F. , Sullivan Patrick A.
Published: July 2002

Recombinant exo‐β‐(1‐3)‐glucanase from Candida albicans was expressed in Saccharomyces cerevisiae and purified. The enzyme contains a number of short blocks of sequence homology with several genes for cellulases of the family A glycanases including the conserved sequence motif NEP which has previously been shown to be important in the catalytic function of several cellulases. Site directed mutagenesis of this glutamic acid residue in the 1,3 glucanase (E230D, E230Q) decreased the enzymatic activity 15,000‐ and 400‐fold, respectively. This suggests that the E of the NEP participates in catalysis of the exoglucanase and other related glycanases.
Genomic DNA structure of a gene encoding cytosolic ascorbate peroxidase from Arabidopsis thaliana
Authors: Kubo Akihiro , Saji Hikaru , Tanaka Kiyoshi , Kondo Noriaki
Published: July 2002

A genomic DNA clone encoding cytosolic ascorbate peroxidase was isolated from a genomic library of Arabidopsis thaliana, using a cDNA for the enzyme as a probe. Nucleotide sequence and primer extension analyses of this gene (APX1) revealed nine exons split by eight introns, one of which is inserted in the 5'‐untranslated region. The exon/intron organization of the APX1 gene differs from that of the guaiacol peroxidase genes.
Evidence for a Rieske‐type FeS center in the thermoacidophilic archaebacterium Sulfolobus acidocaldarius
Authors: Anemüller Stefan , Schmidt Christian L. , Schäfer Günter , Teixeira Miguel
Published: July 2002

A high‐potential iron‐sulfur cluster with characteristics similar to a Rieske‐type center was detected in the plasma membrane of Sulfolobus acidocaldarius by EPR spectroscopy. In the reduced form this center has g‐values of g
z = 2.031, g
y = 1.890 and g
x = 1.725 (g
av = 1.88, rhombicity = 0.37) and its reduction potential at pH 7.4 was determined to be +325 ± 10 mV. The archaebacterial cluster exhibits some unique properties, in comparison to eubacterial and eukaryotic Rieske‐type centers. First, the reduction potential is pH‐dependent in the range from pH 6.7 to 8.2. Second, the typical inhibitor of Rieske FeS centers, DBMIB, had no effect on the g‐values of this cluster. The center is reducible by both NADH and succinate in the presence of cyanide, an inhibitor of the terminal oxidases. The possible role of a Rieske‐type center in an organism lacking any c‐type cytochromes is discussed.
Presence of a novel primary response gene ST2L, encoding a product highly similar to the interleukin 1 receptor type 1
Authors: Yanagisawa Ken , Takagi Toshimitsu , Tsukamoto Toshihiko , Tetsuka Tsunao , Tominaga Shin-ichi
Published: July 2002

In the course of studying the ST2 gene, which was initially found to be expressed specifically at the G0/G1 transitional state in BALB/C‐3T3 cells and was one of the primary response genes, we found another ST2‐related mRNA, designated as ST2L, in serum‐stimulated BALB/c‐3T3 cells in the presence of cycloheximide. Nucleotide sequence analysis of the cloned ST2L cDNA revealed that it had an open reading frame encoding a polypeptide of 567 amino acids. A 5' region (1,028 nucleotides) of ST2L cDNA was identical with the ST2 cDNA, and a unique 3' region encoded a putative transmembrane domain of 24 amino acids and a cytoplasmic domain of 201 amino acids. The ST2 gene product is highly similar to the extracellular portion of IL‐1 receptors type 1 and type 2, and the ST2L gene product shows a marked similarity with entire IL‐1 receptor type 1.
Spectral hole burning study of intact cells of green bacterium Chlorobium limicola
Authors: Fetisova Z.G. , Mauring K.
Published: July 2002

Spectral hole burning studies of intact cells of the green bacterium, Chlorobium limicola, have proven that the Qy‐absorption system of antenna bacteriochlorophyll c (BChl c) should be interpreted in terms of the delocalized exciton level structure of an oligomer. For the first time the 0‐0 band of the lowest exciton state of BChl c oligomers has been directly detected as the lowest energy inhomogeneously broadened band (FWHM −100 cm−1; position of maximum, at ~774 nm) of the near‐infrared BChl c band of 1.8K excitation spectrum (FWHM = 830 cm−1; position of maximum, at 751 nm).
Potentiation of diacylglycerol‐induced activation of protein kinase C by lysophospholipids
Authors: Sasaki Yukio , Asaoka Yoshinori , Nishizuka Yasutomi
Published: July 2002

Lysophospholipid, particularly 2‐lysophosphatidylcholine (lysoPtdCho), significantly potentiates the diacylglycerol (DAG)‐induced activation of protein kinase C (PKC) in vitro. LysoPtdCho shows no effect, unless DAG and phosphatidylserine (PtdSer) are present. This lysoPtdCho action also depends on its own as well as on Ca2+ concentration. At physiological Ca2+ concentrations, the activation of the α‐, β‐, and γ‐subspecies (cPKC) is enhanced by lysoPtdCho in the 10−6 M range, but inversely inhibited in the 10−5 M range. The δ‐ and ε‐subspecies (nPKC), which are enzymatically insensitive to Ca2+, are mostly inhibited by lysoPtdCho at its low concentrations. The enhancement of cPKC activation by lysoPtdCho is due to the increase in an apparent affinity of the enzyme for PtdSer but not for DAG. The results may account, at least in part, for the previous observations made with intact cell systems that lysoPtdCho significantly potentiates the DAG‐induced cellular responses such as T‐lymphocyte activation and HL‐60 cell differentiation [(1992) Trends Biochem. Sci. 17, 414–4171].
The interaction of metal ions with annexin V: A crystallographic study
Authors: Lewit-Bentley Anita , Bentley Graham Arthur , Favier Béatrice , L'Hermite Guillaume , Renouard Madalena
Published: July 2002

Three closely related rhombohedral crystal structures of human annexin V have been analysed and compared: a low‐calcium, a high‐calcium and an ytterbium‐soaked crystal. The occupancy of the calcium sites increases at higher calcium concentrations, but the calcium is removed rather than replaced during soaking in the ytterbium solution. Instead, other sites are substituted at high calcium concentrations as well as in the presence of ytterbium. Furthermore, a new site is revealed in the ytterbium‐soaked crystal which may give a clue to the mechanism of conformational change that takes place in the third domain of annexin V in the presence of very high calcium concentrations and of phospholipids.
Differential effects of fatty acid and phosholipid activators on the catalytic activities of a structurally novel protein kinase from rat liver
Authors: Morrice Nicholas A. , Fecondo John , Wettenhall hard E.H.
Published: July 2002

The lipid responsiveness of the structurally unique protein kinase, referred to as PAK‐1, recently isolated from rat liver [(1994) J. Biol. Chem. 269, in press], is characterised by the high sensitivity (low micromolar) of its ribosomal S6(229–239) peptide kinase activity to both cardiolipin and the cis‐unsaturated fatty acids and insensitivity to phosphatidylserine. Autophosphorylation of PAK‐1 exhibited even greater sensitivity (submicromolar) to cardiolipin, but was relatively less affected by phosphatidylserine. Oleate, the most potent activator of PAK‐1's peptide kinase activity was relatively ineffectual with autophosphorylation. These and other unusual characteristics, including high levels of basal catalytic activities, suggest a novel mechanism of regulation distinct from that of the protein kinase Cs
A 70‐kDa heat shock cognate protein suppresses the defects caused by a proteasome mutation in Saccharomyces cerevisiae
Authors: Masayuki Ohba
Published: July 2002

An allele of mutation in the proteasome subunit gene Y7, y7‐1, caused a temperature‐sensitive growth in S. cerevisiae. One of the multi‐copy suppressor genes for this growth defect was identical to SSB1, which encodes a 70‐kDa heat shock cognate protein of the yeast. Introduction of the multi‐copy SSB1 gene into the y7‐1 mutant cells suppressed defects in the degradation of X‐β‐galactosidase (X = Arg or Pro) observed in the mutant cells. Thus, the SSB1 protein, one of the chaperons of the yeast, facilitated intracellular protein degradation.
cDNA cloning of a cytosolic protein tyrosine phosphatase (RKPTP) from rat kidney
Authors: Moriyama Toshiki , Kawanishi Sachio , Inoue Takuya , Imai Enyu , Kaneko Tetsuya , Xia Chen , Takenaka Masaru , Noguchi Tamio , Kamada Takenobu , Ueda Naohiko
Published: July 2002

A rat cDNA encoding a non‐receptor type phosphotyrosine phosphatase (PTPase; EC 3.1.3.48) was identified. The 1608 bp cDNA contains a single open reading frame that predicts a 382 amino acid protein with M
r 44,438. The predicted protein has no apparent signal or transmembrane sequences, suggesting that it is a cytosolic protein. The C‐terminal region has a PTPase catalytic domain that has 40–50% nucleic acid homology to other known PTPases. The N‐terminal region has little amino acid sequence homology to any other known sequences. The recombinant protein of the cloned cDNA expressed in Escherichia coli was shown to possess PTPase activity using myelin basic protein, tyrosine phosphorylated by p43ν‐abl
tyrosine kinase, as a substrate.
A subtype of the metabotropic glutamate receptor family in the olfactory system of Atlantic salmon
Authors: Pang Jiongdong , Lo Ying Har , Chandlee Joel M. , Rhoads Dennis E.
Published: July 2002

A plasma membrane rich fraction was prepared from olfactory rosettes of Atlantic salmon and used to study binding of l‐glutamic acid and activation of phospholipase C (PLC). Glutamate binding was saturable, high affinity, and inhibited by aspartic acid and taurocholate but not by alanine and lysine. Binding of glutamate was potently inhibited by various ligands for rat brain metabotropic glutamate receptors (mGluR) and also by kainate and N‐methyl‐d‐aspartate. Glutamate stimulated phosphatidylinositol 4,5‐bisphosphate breakdown consistent with G protein‐dependent activation of PLC. Northern blot analyses demonstrated the presence of olfactory rosette RNA that hybridizes with cDNA probes for mGluR1 and mGluR4 under low stringency conditions. The results indicate the salmon olfactory system includes a subtype of the metabotropic glutamate receptor family.
Common structural motifs in small proteins and domains
Authors: Efimov A.V
Published: July 2002

The high frequency of occurrence of the definite folding units in unrelated proteins and the fact that many small proteins are merely composed of the folding units indicate that these units can fold into unique structures per se and can be nuclei or ‘ready building blocks’ in protein folding.
New insights into the auxiliary domains of eukaryotic RNA binding proteins
Authors: Biamonti Giuseppe , Riva Silvano
Published: July 2002

Eukaryotic RNA binding proteins (RBP) are key players in RNA processing and in post‐transcriptional regulation of gene expression. By interacting with RNA and other factors and by modulating the RNA structure, they promote the assembly of a great variety of specific ribonucleoprotein complexes. Many RBPs are composed of highly structured and conserved RNA binding domains (RBD) linked to unstructured and divergent auxiliary domains; such modular structure can account for a multiplicity of interactions. In this context, the auxiliary domains emerge as essential partners of the RBDs in both RNA binding and functional specialisation. Moreover, the determinants of biologically important functions, such as strand annealing, protein‐protein interactions, nuclear localization and activity in in vitro splicing, seem to reside in the auxiliary domains. The structural and functional properties of these domains suggest their possible derivation from ancestral non‐specific RNA binding polypeptides.
A phorbol ester‐responsive PKC‐ζ generated by fusion with the regulatory domain of PKC‐δ
Authors: Goode Nigel T. , Parker Peter J.
Published: July 2002

A hybrid molecule generated by fusing the regulatory domain of PKC‐δ with the catalytic domain of PKC‐ζ is, like PKC‐δ but unlike PKC‐ζ, a phorbol ester‐dependent enzyme. However, the substrate specificity of this hybrid resembles that of PKC‐ζ. Expression of mammalian PKC‐δ, but not PKC‐ζ, m the fission yeast Schizosaccharomyces pombe causes growth retardation and phorbol esters amplify the PKC‐δ phenotype without affecting that of PKC‐ζ (Goode et al., submitted). The chimaeric molecule also inhibited growth and this effect was phorbol‐ester dependent. Both the hybrid and PKC‐δ holoenzyme, in contrast to PKC‐ζ, down‐regulate upon prolonged exposure to phorbol esters in vivo. Thus, this hybrid retains the regulatory properties conferred by PKC‐δ but the catalytic properties of PKC‐ζ. This regulatable chimaeric molecule will be useful in assessing the function of PKC‐ζ.
Furin has the proalbumin substrate specificity and serpin inhibitory properties of an in situ hepatic convertase
Authors: Brennan Stephen O. , Nakayama Kazuhisa
Published: July 2002

Furin, a KEX2 protease homolog with high RNA expression in the liver is an excellent candidate as a hepatic proprotein convertase. Here we show that purified recombinant furin has the same proalbumin specificity and serpin inhibitory properties as the in situ hepatic convertase. There was rapid cleavage at the ‐RRD‐ site of normal human proalbumin but there no significant cleavage of natural unprocessed variants with cleavage site sequences of ‐RRV‐, ‐HRD‐, ‐RQD‐, or ‐CRD‐. Cleavage of the latter was not increased by S‐aminoethylation. Furin was specifically inhibited by α1‐antitrypsin Pittsburgh (358 Met → Arg), (K
= 3 μM) but not by 50 μM normal antitrypsin M or by antithrombin, however, antithrombin/heparin was a good inhibitor (K
= 9 μM). The pH optimum for proalbumin cleavage was between pH 5.5 and 6.0, indicating that furin is potentially fully active within secretory vesicles, the site of proalbumin cleavage.
Stereochemical constraint in the evolution of pyridoxal‐5′‐phosphate‐DApenDAnt enzymes. A hypothesis
Authors: Christen Philipp , Kasper Patrik , Gehring Heinz , Sterk Michael
Published: July 2002

In the transamination reactions undergone by pyridoxal‐5′‐phosphate‐DApenDAnt enzymes that act on l‐amino acids, the C4′ atom of the cofactor is without exception protonated from the si siDA. This invariant absolute stereochemistry of enzymes not all of which are evolutionarily related to each other and the inverse stereochemistry in the case of d‐alanine aminotransferase might reflect a stereochemical constraint in the evolution of these enzymes rather than an acciDAntal historical trait passed on from a common ancestor enzyme. Conceivably, the coenzyme and substrate binding sites of primordial pyridoxal‐5′‐phosphate‐DApenDAnt enzymes had to fulfil the following prerequisites in order to allow their DAvelopment toward effective catalysts: (i) the negatively charged α‐carboxylate group of the amino acid substrate had to be positioned as far as possible away from the negatively charged phosphate group of the cofactor, and (ii) the Cα‐H bond had to be oriented toward the protein. Compliance with these two criteria implies, under the assumption that C4′ is protonated by an acid‐base group of the protein, the observed stereochemical feature.
Spermatocyte‐specific expression of the gene for mouse testis‐specific transcription elongation factor S‐II
Authors: Ito Takahiro , Xu Qihong , Takeuchi Hideaki , Kubo Takeo , Natori Shunji
Published: July 2002

Previously, we characterized a rat cDNA for testisspecific transcription elongation factor S‐II (SII‐T1) (Q. Xu et al., J. Biol. Chem. 269, 3100–3103 (1994)). Here, we isolated a 335‐bp fragment of the cDNA for mouse SII‐T1, and used it to examine the expression of the SII‐T1 gene in the testis by in situ hybridization. The results indicated that the SII‐T1 gene is expressed exclusively in spermatocytes, showing no appreciable expression in spermatogonia, spermatids, or Leydig cells. RTPCR experiments using testis RNA from WI Wv mutant mice also suggested that SII‐T1 is a specific transcription elongation factor essential for spermatogenesis.
The Saccharomyces cerevisiae kinetochore
Authors: Lechner Johannes , Ortiz Jennifer
Published: July 2002

Accurate chromosome segregation is DApenDAnt on a specialized chromosomal structure, the kinetochore/centromere. The only essential constituent of the S. cerevisiae kinetochore established today is CBF3, a multisubunit complex that binds to S. cerevisiae centromere DNA. Therefore CBF3 and its four components, Cbf3a, Cbf3b, Cbf3c and Cbf3d, will form the centerpiece of this review. In addition, we will DAscribe proteins that are putatively involved in kinetochore function specifically in the context with CBF3 interaction. Furthermore, we discuss the role of the S. cerevisiae kinetochores in a putative cell cycle checkpoint control and in microtubule attachment.
Transmembrane segment 10 is important for substrate recognition in Ga12 and Hxt2 sugar transporters in the yeast Saccharomyces cerevisiae
Authors: Kasahara Michihiro , Shimoda Eriko , Maeda Mari
Published: July 2002

A systematic series of chimeras between Gal2 galactose transporter and Hxt2 glucose transporter in yeast was produced to delineate the essential domain for substrate recognition. A domain of 101 amino acids close to the COOH‐terminus that has been previously identified as the critics substrate recognition region was further divided into four subdomains, by introducing five restriction enzyme sites at exactly corresponding locations of both genes without changing coding amino acids. When each of all possible 16 modified genes was expressed, all the galactose transport‐active chimeras were found to possess Ga12‐derived transmembrane segment (TM) 10. Of the 35 amino acids in the TM10 region, only 12 differ between Ga12 and Hxt2, indicating that these 12 amino acids include the critical residue(s) responsible for the differential recognition of galactose and glucose in these transporters.
The type‐1 repeats of thyroglobulin regulate thyroglobulin degradation and T3, T4 release in thyrocytes
Authors: Molina Franck , Pau Bernard , Granier Claude
Published: July 2002

Thyroglobulin (Tg) proteolytic steps are central phenomena in Tg processing and thyroid hormone release in thyrocytes. Based on recent literature data, we propose that the type‐1 repetitive units present in the Tg sequence could act as binders and reversible inhibitors of the proteases implicated in Tg processing. The pH‐dependent interactions of proteases with the repeats could permit (i) protection from degradation of low iodinated Tg to be recycled; (ii) restriction of early proteolytic attacks to N‐ and C‐terminal hormone formation sites; (iii) increase of the half‐time of acidic proteases necessary for the final, extensive degradation steps of Tg.
rab3‐Peptide stimulates exocytosis of the ram sperm acrosome via interaction with cyclic AMP and phospholipase A2 metabolites
Authors: Garde J. , Roldan E.R.S.
Published: July 2002

Acrosomal exocytosis triggered with A23187/Ca2+ was enhanced by rab3AL, a synthetic peptide corresponding to the effector domain of the small GTP‐binding protein rab3. Exocytosis was further enhanced when spermatozoa were also exposed to dibutyryl‐cAMP, but was prevented when H‐89, a protein kinase A (PKA) inhibitor, was included. The action of rab3AL was not on, or upstream of, phospholipase A2 (PLA2). Inhibition of exocytosis by the PLA2 inhibitor aristolochic acid was overcome by rab3AL when it was included together with lysophosphatidylcholine; this effect was prevented by H‐89. These results suggest a functional coupling between rab3 protein, metabolites generated by PLA3, and cAMP‐activated PKA, in the final steps leading to membrane fusion during acrosomal exocytosis.