bluuebunny/crossref_metadata_2025 huggingface dataset:
Two changes of the same nucleotide confer resistance to diuron and antimycin in the mitochondrial cytochrome b gene of Schizosaccharomyces pombe
Authors: Weber S. , Wolf K.
Published: July 2002

Diuron (3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea) and antimycin, both inhibitors of mitochondrial respiration, block electron flow between cytochromes b and c
1. Mutants resistant to either drug have been selected using Schizosaccharomyces pombe strains with an extrachromosomally inherited mutator. In analogy to Saccharomyces cerevisiae these mutational sites were assumed to map in the cytochrome b gene. DNA sequence analysis showed that two changes in the same nucleotide are responsible for resistance to antimycin and diuron. Analysis of resistant and sensitive progeny of crosses between the mutants and the wild type confirmed the correlation between mutational alteration and resistant phenotype.
The testicular transcript of the angiotensin I‐converting enzyme encodes for the ancestral, non‐duplicated form of the enzyme
Authors: Lattion Anne-Laure , Soubrier Florent , Allegrini Jacqueline , Hubert Christine , Corvol Pierre , Alhenc-Gelas François
Published: July 2002

The endothelial angiotensin I‐converting enzyme (ACE) is organized in two large homologous domains, each bearing a putative active site. However, only one of these sites is probably involved in catalysing the conversion of angiotensin I into angiotensin II. The testicular form of ACE is equally active, encoded by the same gene, but translated from a shorter mRNA. Molecular cloning of the human testicular ACE cDNA indicates that the mRNA codes for 732 residues (vs 1306 in endothelium). The testicular transcript corresponds to the 3′ half of the endothelial transcript and encodes one of the two homologous domains of endothelial ACE, preceded by a short specific sequence. This 5′ specific sequence contains 228 nucleotides and encodes 67 amino acids, including the putative signal peptide followed by a serine/threonine‐enriched region, presumably glycosylated. The testicular transcript corresponds to the ancestral, non‐duplicated form of the ACE gene. Since the carboxyl‐terminal domain of the endothelial ACE is expressed in the testicular enzyme, it is likely that it bears the active site in both forms.
Molecular cloning and nucleotide sequence analysis of mRNA for human kidney ornithine aminotransferase
Authors: Kobayashi Tatsuhiko , Nishii Masatoshi , Takagi Yasuyuki , Titani Koichi , Matsuzawa Takeo
Published: July 2002

The cDNA encoding ornithine aminotransferase (EC 2.6.1.13; OAT) was isolated from a human kidney cDNA library. The isolated cDNA contained the entire protein coding region and partial 3′‐ and 5′‐untranslated regions. The nucleotide sequences of human kidney OAT cDNA were absolutely homologous with those of human liver OAT cDNA, and human kidney and liver OAT fused completely against the antibody to human kidney OAT in an Ouchterlony double diffusion test. These findings settled the controversy as to which characteristics of liver and kidney OAT isozymes are different. An N‐terminal sequence analysis of purified mature human kidney OAT clarified that the leader peptide was cleaved between Gln‐35 and Gly‐36.
Reversible modulation of the mitochondrial ATP synthase with energy demand in cultured rat cardiomyocytes
Authors: Das Anibh M. , Harris David A.
Published: July 2002

Mitochondria; Cardiomyocyte; ATP synthase; ATPase inhibitor protein; Metabolic regulation; Anoxia
An oligonucleotide hybridization approach to DNA sequencing
Authors: Khrapko K.R. , P. Lysov Yu , Khorlyn A.A. , Shick V.V. , Florentiev V.L. , Mirzabekov A.D.
Published: July 2002

DNA sequencing; Oligonucleotide hybridization, immobilized
The prosomal protein of 27 kDa and a nuclear 38 kDa protein are immunologically related
Authors: Kreutzer-Schmid Claudia , Schmid Hans-Peter
Published: July 2002

A monoclonal antibody, recognizing the prosomal 27 kDa protein, was used to detect prosomal proteins in nuclear fractions. Protein gel electrophoresis and immunoblotting revealed large amounts of a 38 kDa protein and in very low amount a 27 kDa protein reacting with the monoclonal antibody, Immunoblot analysis of V8 protcolytic fragments demonstrated that both proteins arc intimately related and suggest that the nuclear 38 kDa protein could be a precursor of the 27 kDa protein (pros P 27) which is a structural component of cytoplasmic prosomes.
Oxidation of coniferyl alcohol by cell wall peroxidases at the expense of indole‐3‐acetic acid and O2
Authors: Ferrer María A. , Pedreño María A. , Muñoz Romualdo , Barceló A.Ros
Published: July 2002

The oxidation of coniferyl alcohol (CA), a lignin precursor, by cell wall peroxidases may take place at the expense of indole‐3‐acetic acid (IAA) and O2, and in the absence of H2O2. The peroxidase‐catalyzed oxidation of CA shows an optimum at an IAA concentration of 0.33 mM, while higher IAA concentrations are inhibitory. The observation that the oxidation of CA by cell wall peroxidase at the expense of IAA and O2 is inhibited by genistein, a putative endogenous inhibitor of lignification in lupin hypocotyls, supports the view that the H2O2‐generating system coexists with cell wall peroxidase activities involved in lignification, and that it takes place at the expense of IAA and O2.
The ammonium sulfate activation of phosphorylase b
Authors: Leonidas D.D. , Oikonomakos N.G. , Papageorgiou A.C. , Xenakis A. , Cazianis C.T. , Bem F.
Published: July 2002

The ammonium sulfate activation of phosphorylase b has been studied. Ammonium sulfate, when present in high concentrations, induces properties of phosphorylase a in phosphorylase b, such as an enhanced affinity for AMP, a reversal of the glucose‐6‐P inhibition and enzyme tetramerization. The data are consistent with the interpretation that sulfates bind to the Ser‐14 site and the sulfate‐protein interactions at this site are responsible for activation of phosphorylase b.
Structural kinetics of the allosteric transition of aspartate transcarbamylase produced by physiological substrates
Authors: Tsuruta Hirotsugu , Sano Takayuki , Vachette Patrice , Taue Patrick , Moody Michael F. , Wakabayashi Katsuzo , Amemiya Yoshiyuki , Kimura Kazumoto , Kihara Hiroshi
Published: July 2002

We have studied the kinetics of the quaternary structure change associated with the allosteric transition of aspartate transcarbamylase (ATCase) (E. coli), inducing this change by exposure to the natural substrates (carbarnyl phosphate and L‐aspartate). The presence of 30% ethylene glycol slowed the quaternary structure change sufficiently for it to be followed by stopped‐flow X‐ray scattering at − 5°C. After adding substrates to the enzyme, the change occurred, with a half‐life of a few seconds, yielding a mixture of the two standard quaternary structures (or, conceivably, a state intermediate between them). This mixture persisted until the enzyme reduced the substrate concentration below a threshold value.
Ceramidase activity in porcine epidermis
Authors: Wertz Philip W. , Downing Donald T.
Published: July 2002

This report presents the first demonstration of a ceramide‐hydrolyzing activity in mammalian epidermis. An assay using fractions derived from porcine epidermis and synthetic [3H]ceramide is described, and it is shown that under the conditions used, the K
m for ceramide is 110 μM and hydrolysis is linear for up to 2 h. The enzyme activity is maximal at pH 8‐9. The specific activity of ceramide hydrolase decreases as the protein concentration in the assay mixture increases, suggesting the possibility of a dissociable inhibitor. Also, the activity can be inhibited by added palmitic acid. Ceramide hydrolysis may be an important regulatory mechanism in the epidermis due to the ability of the liberated free sphingosine to modulate the activity of protein kinase C.
Identification of phosphopeptides by mass spectrometry
Authors: Schroeder Werner , Covey Thomas , Hucho Ferdinand
Published: July 2002

Phosphopeptides can be identified by ion spray mass spectrometry. The method was tested with phosphokemptide and with a proteolytic digest of one subunit (δ‐subunit) of the nicotinic acetylcholine receptor. In the latter one peptide containing tyrosine phosphate, one with two serine phosphates, and two different peptides each containing one serine phosphate were unambiguously identified. Thus it is proven that ion spray mass spectrometry can be applied for the localization of phosphorylation sites in a known primary structure.
Plant and fungus calmodulins are polyubiquitinated at a single site in a Ca2+‐dependent manner
Authors: Ziegenhagen R. , Jennissen H.P.
Published: July 2002

In plants Ca2+ plays a crucial role as second messenger. Thus calmodulin is one of the most important signal transducing molecules for metabolic regulation in plants. Previously we showed that bovine testis calmodulin can be covalently coupled at one site to ubiquitin in a Ca2+‐dependent manner in the presence of ATP/Mg2+ by ubiquityl‐calmodulin synthetase. Since calmodulin from spinach has 13 amino acid sequence differences to bovine calmodulin ‐ two of them in Ca2+‐binding loops ‐ it was unclear, whether a conjugation of ubiquitin to this molecule would be possible. In this paper it is shown that calmodulin from spinach and a similar calmodulin from the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca2+‐dependent manner. It is shown that higher molecular mass conjugates containing up to three ubiquitin molecules per calmodulin are obtained. Experiments with methylated ubiquitin demonstrate that, as with vertebrate calmodulins, only one lysine residue is linked to ubiquitin and that the incorporation of addditional ubiquitin molecules leads to a polyubiquitin chain.
Lysophosphatidylcholine specifically stimulates plasma membrane H+‐ATPase from corn roots
Authors: Pedchenko V.K. , Nasirova G.F. , Palladina T.A.
Published: July 2002

The plasma membrane H+‐ATPase activity from corn seedling roots is shown to be stimulated 3‐ to 4‐fold by the addition of lysophosphatidylcholine (lysoPC). This effect clearly differs from that of other detergents by both the magnitude and the absence of inhibition at higher concentrations. LysoPC decreases the apparent K
m for MgATP, increases V
max of the ATPase reaction but does not change its pH optimum. On the contrary, the acid phosphatase activity associated with plasma membranes is not influenced by lysoPC. A lysoPC stimulation is also demonstrated for the solubilized preparation of the H+‐ATPase. It is assumed that lysoPC stimulation of the plant plasma membrane H+‐ATPase is not only due to permeabilization of the vesicles for MgATP, but also direct action on the enzyme.
Chlorophyll levels in the pigment‐binding proteins of photosystem II A study based on the chlorophyll to cytochrome ratio in different photosystem II preparations
Authors: Barbato Roberto , Race Helen L. , Friso Giulia , Barber James
Published: July 2002

The chlorophyll levels in pigment proteins of photosystem II were investigated by using photosystem II preparations with different levels or complexity. Based on the assumption that there is I cytochrome b559 per reaction centre it has been found that oxygen‐evolving complexes containing CP26 and CP29 bind 42 chlorophyll molecules, When CP26 and CP29 are stripped away, the resulting PSII cores bind 30 chlorophyll molecules while CP43‐less cores bind approximately 18 chlorophylls. It is therefore concluded that CP47 and CP43 bind 9–12 molecules of chlorophyll a and the D1/D2 complex binds 6 chlorophylls. Taken together CP26 and CP29 bind about 12 chlorophyll molecules.
Effects of substitutions of glycine and asparagine for serine132 on activity and binding of human lipoprotein lipase to very low density lipoproteins
Authors: Tashiro Jun , Kobayashi Junji , Shirai Kohji , Saito Yasushi , Fukamachi Isamu , Hashimoto Hideyuki , Nishida Tsutomu , Shibui Tatsuro , Morimoto Yuuki , Yoshida Sho
Published: July 2002

For studying the role of Ser132 in the putative catalytic site of human lipoprotein lipase (LPL), mutant LPL cDNAs expressing LPLs with amino acid substitutions of Gly or Asn for Ser132 were obtained by site‐directed mutagenesis, and were expressed in COS‐1 cells. Considerable amounts of LPL enzyme protein mass were detected in the culture medium of COS‐1 cells transfected with wild‐type LPL, LPL‐Gly132, or LPL‐Asn132. LPL‐Gly132 hydrolyzed Triton X‐100‐triolein and tributyrin as effectively as wild‐type LPL, whereas LPL‐Asn132 showed no activity. LPL‐Asn132 bound to very low density lipoproteins as effectively as wild‐type LPL.
The application of 1H NMR chemical shift calculations to diastereotopic groups in proteins
Authors: Williamson Michael P. , Asakura Tetsuo
Published: July 2002

We have calculated chemical shifts for a range of diastereotopic protons in proteins (i.e. methylene protons, and the methyl groups of valine and leucine residues), using a recently optimised method for chemical shift calculation. The calculations are based on crystal structure coordinates, and ave been compared with experimental stereospecific assignments. The results indicate that chemical shifts can be used to suggest stereospecific assignments with about 80% probability of being correct, in cases where both the experimental and the calculated chemical shift differences between a pair of diastereotopic protons are greater than 0.3 ppm. Inaccurate calculations are shown to be caused in most cases by differences between crystal and solution structures. Furthermore, chemical shift calculations based on NMR structures are shown to be capable of acting as a further constraint on structure, by limiting the range of side‐chain conformations adopted in structures calculated from NMR data.
Correlation between local cell membrane displacements and filterability of human red blood cells
Authors: Tuvia Shmuel , Levin Shlomo , Korenstein Rafi
Published: July 2002

Local mechanical fluctuations of the cell membrane of human erythrocytes were shown to involve MgATP‐ and Mg2+‐driven fast membrane displacements. We propose that these local bending deformations of the cell membrane are important for cell passage through capillaries. In order to verify this hypothesis, we examined cell membrane fluctuations and filterability of erythrocytes over a wide range of medium osmolalities (180–675 mosmol/kg H2O). The results indicate the existence of a positive correlation between the amplitude of local cell membrane displacements and cell filterability. We suggest that the occurrence of metabolically driven membrane displacements on the side surface of the red blood cell diminishes its bending stiffness and enables it to fold more efficiently upon entrance into blood capillaries. Thus, local cell membrane displacements seem to play an important role in microcirculation.
Photoinduced degradation of the D1 protein in isolated thylakoids and various photosystem II particles after donor‐side inactivations Detection of a C‐terminal 16 kDa fragment
Authors: Barbato Roberto , Frizzo Annalisa , Friso Giulia , Rigoni Fernanda , Giacometti Giorgio Mario
Published: July 2002

Photoinduced degradation of the photosystem II (PSII) reaction center D1 protein was studied in isolated thylakoids and different PSII subparticles. A 16 kDa fragment corresponding to the C‐terminus of the protein is detected in thylakoids when they are inactivated at the donor side before illumination. The same D1 fragment is found in different types of PSII preparations at different integration levels characterized by different polypeptide compositions so long as they have an inactivated donor side and an active electron acceptor for the reduced pheophytin. However, when the PSII particle is equal to or smaller than the 43‐less PSII core complex, other fragments are observed which are not found in more integrated systems.
Natural polypeptides in left‐handed helical conformation A circular dichroism study of the linker histones’ C‐terminal fragments and β‐endorphin
Authors: Makarov A.A. , Lobachov V.M. , Adzhubei I.A. , Esipova N.G.
Published: July 2002

Circular dichroism has been used to investigate the histone HI and H5 C‐terminal fragments and β‐endorphin conformation. It has been shown that in aqueous solution these polypeptides preferably adopt the left‐handed helical conformation of the poly‐l‐proline II type. A break in the linear temperature dependence of the CD value was found in the temperature interval between 50 and 55°C. It was proposed to be due to non‐cooperative disordering of the conformation caused by the destruction of the hydration shell.
H+‐coupled (Na+‐independent) proline transport in human intestinal (Caco‐2) epithelial cell monolayers
Authors: Thwaites David T. , McEwan Gordon T.A. , Cook Martin J. , Hirst Barry H. , Simmons Nicholas L.
Published: July 2002

Previously, absorption of l‐proline across the apical membrane of the intestinal enterocyte has been attributed to transport via the Na+‐dependent Imino system. However, net (absorptive) transport of proline across intact Caco‐2 cell monolayers was enhanced by acidification of the apical environment, under both Na+‐containing and Na+‐free conditions. This Na+‐independent pH‐dependent proline flux was associated with H+ flow across the apical membrane as determined by continuous measurement of intracellular pH. H+/proline symport was associated with an inward I
sc in voltage‐clamped Caco‐2 epithelial layers demonstrating the electrogenic nature of this transport process. In conclusion Caco‐2 cells possess an apically‐localised, Na+‐independent, electrogenic H+/imino acid transporter which may play an important role in intestinal proline absorption.
Analysis of the role of the COL1 domain and its adjacent cysteine‐containing sequence in the chain assembly of type IX collagen
Authors: Labourdette Lionel , van der Rest Michel
Published: July 2002

The mechanisms of chain selection and assembly of type IX collagen, a heterotrimer α1(IX)α2(IX)α3(IX), must differ from that of fibrillar collagens since it lacks the characteristic C‐propeptide of these latter molecules. We have tested the hypothesis that the information required for this process is contained within the C‐terminal triple helical disulfide‐bonded region (LMW). The reassociations of the purified LMW fragments of pepsinized bovine type IX collagen were followed by the formation of disulfide‐bonded multimers. Our data demonstrate that only three triple helical assemblies form readily, (α1)3, (α2)3, and α1α2α3. The information required for chain selection and assembly is thus, at least in part, contained in the studied fragments. Molecular stoichiometries different from the classical heterotrimer may thus also form under certain conditions.
Internal ribosome entry site ofencephalomyocarditis virus RNA is unable to direct translation in Saccharomyces cerevisiae
Authors: Evstafieva A.G. , Beletsky A.V. , Borovjagin A.V. , Bogdanov A.A.
Published: July 2002

To evaluate the potential of the encephalomyocarditis virus (EMCV) internal ribosome entry site (1RES) to promote efficient expression of foreign genes in the yeast, S. cerevisiae, we have constructed E.coli‐yeast shuttle vectors in which the EMCV 5' non‐coding region was fused to the reporter gene, human prothymosin α. Efficiency of translation of corresponding RNA transcripts in mammalian cell‐free systems was highly dependent on the sequence context and/or position of the initiation codon. No translation of these IRES‐dependent mRNAs occurred in S. cerevisiae.
Participation of the g = 1.9 and g = 1.82 EPR forms of the semiquinone‐iron complex, QA−·Fe2+ of photosystem II in the generation of the Q and C thermoluminescence bands, respectively
Authors: Demeter S. , Goussias Ch. , Bernát G. , Kovács L. , Petrouleas V.
Published: July 2002

Following illumination at 200 K, the charge recombination reactions and the origin of the thermoluminescence (TL) bands appearing at about 0°C (Q band) and +50°C (C band) in the glow curve were investigated by comparative TL and EPR measurements in DCMU‐treated photosystem II particles. Decay half‐time measurements carried out at −25°C and +25°C, respectively, suggest that the S2 state (multi‐line signal) undergoes charge recombination with the g = 1.9 form of the semiquinone‐iron complex, QA
−·Fe2+, resulting in the appearance of the Q band, and that the g = 1.82 form of QA
−·Fe2+ back‐reacts with the oxidized tyrosine, YD
+ (Signal IIs), accounting for the generation of the C band.
Candida parapsilosis expresses and secretes two aspartic proteinases
Authors: Fusek Martin , Smith Elizabeth A. , Monod Michel , Foundling Stephen I.
Published: July 2002

We have isolated and characterized a second aspartic proteinase secreted by the CHUV E‐18 strain of Candida parapsilosis. This proteinase is produced at a level corresponding to approximately 25% of the production of the main proteinase described earlier [1]. This minor proteinase has similar molecular weight and pH optimum but differs in the isoelectric point and in the specificity when compared with the major secreted form. The determination of the amino terminal amino acid sequence identified this minor form of Candida parapsilosis aspartic proteinase as a protein which corresponds to the sequence deduced from genomic DNA originally reported as a pseudogene [1]. We conclude that strain CHUV E‐18 of Candida parapsilosis expresses and secretes two different aspartic proteinases.
Complementation in situ of the yeast plasma membrane H+‐ATPase gene pmal by an H+‐ATPase gene from a heterologous species
Authors: Palmgren Michael Gjedde , Christensen Gertrud
Published: July 2002

In plants and fungi, the transport of solutes across the plasma membrane (pm) is driven by a proton pump (H+‐ATPase) that produces an electric potential and a pH gradient. We expressed AHA2, a member of the Arabidopsis thaliana pm H+‐ATPase gene family, in yeast cells in which transcription of the endogenous pm H+ATPase gene (pmal) had been turned off. AHA2 was expressed mainly in intracellular membranes and only supported very slow growth of transformed yeast cells. Removal of the last 92 C‐terminal amino acids from the plant H+‐ATPase produced an enzyme with 2–3‐fold higher specific ATPase activity than the wild‐type plant enzyme. Surprisingly, the truncated H+‐ATPase was now targetted to the yeast pm and fully supported normal yeast growth.
Analysis of the catalytic center of Cyclomaltodextrinase from Thermoanaerobacter ethanolicus 39E
Authors: Podkovyrov Sergey M. , Burdette Douglas , Zeikus J.Gregory
Published: July 2002

The amino acid sequences of cyclomaltodextrinase (CDase) from Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E) and other amylolytic enzymes were compared by using linear alignment and hydrophobic cluster analysis. Two Asp and one Glu residue, which were considered to be the catalytic residues of the compared enzymes according to crystallographic or protein engineering experiments, were also conserved in CDase. Asp325, Asp421 and Glu354 of the CDase were individually replaced by means of site‐directed mutagenesis. The mutant enzymes completely lost activity, suggesting that these residues play an important role in catalysis.
N‐Peptidyl‐O‐carbamoyl amino acid hydroxamates: Irreversible inhibitors for the study of the S2′ specificity of cysteine proteinases
Authors: Brömme Dieter , Kirschke Heidrun
Published: July 2002

A series of new inhibitors for cysteine proteinases with the general structure Z‐Phe‐Gly‐NHO‐CO‐Aa (Aa = amino acids) was synthesized and tested as inhibitors of papain‐like enzymes (cathepsins S, L, B and papain). Like N‐peptidyl‐O‐acyl hydroxamates the inhibitors inactivate cysteine proteinases by a sulfenamidation of the active site cysteine residue. The most effective inhibitors display second order‐rate constants of inactivation in the range of 103–104 M−1·s−1. Since the structure of the N‐peptidyl‐O‐carbamoyl amino acid hydroxamates allows the variation of the leaving group this class of inhibitors was used as a new tool for the evaluation of the S2′ specificity of cysteine proteinases.
Cloning and expression in yeast of a higher plant chorismate mutase Molecular cloning, sequencing of the cDNA and characterization of theArabidopsis thalianaenzyme expressed in yeast
Authors: Eberhard Jenny , Raesecke Hanns-R. , Schmid Jürg , Amrhein Nikolaus
Published: July 2002

Chorismate mutase (EC 5.4.99.5) catalyzes the first step in the branch of the shikimate pathway which leads to the aromatic amino acids, phenylalanine and tyrosine. We have isolated a cDNA for this enzyme from the higher plant,Arabidopsis thaliana, by complementing a yeast strain (aro7) with a cDNA library fromA. thaliana. This is the first chorismate mutase cDNA isolated from a plant. It encodes a protein of 334 amino acids. The identity of the deduced amino acid sequence is 41% to the chorismate mutase sequence fromSaccharomyces cerevisiae. The N‐terminal portion of the deduced amino acid sequence has no homology to theS. cerevisiaesequence but resembles known plastid‐specific transit peptides. TheA. thalianachorismate mutase expressed in yeast revealed allosteric control by the three aromatic amino acids, as previously described for plastidic chorismate mutase isozymes.
Selective nuclear translocation of protein kinase C α in Swiss 3T3 cells treated with IGF‐I, PDGF and EGF
Authors: Neri Luca M. , Billi Anna Maria , Manzoli Lucia , Rubbini Silvia , Gilmour R.Stewart , Cocco Lucio , Martelli Alberto M.
Published: July 2002

To determine the subcellular distribution of PKC after GFs treatment we have employed a combined immunochemical and in situ confocal microscopy analysis. In quiescent SvMs 3T3 cells only a faint PKC positivity was observable in the nucleus while a strong reaction was seen in the cytoplasm. IGF‐I and to a lesser extent PDGF and EGF induced, after 45 min of treatment, a nuclear translocation of PKC detected by a pan‐anti‐PKC antibody and nuclear fluorescence was distributed in the nuclear interior except for the nucleolar regions. Bombesin and FGF did not affect the sub‐cellular distribution of the enzyme. To further the understanding of which PKC isoform was involved in the transiocation process, we have tested nine isozyme‐speeffic anti‐PKC antibodies. Immunoblotting analysis revealed the presence in Swiss 3T3 fibroblasts of α, βI, ε and ζ isoforms. In isolated nuclei from GF‐exposed cells only the α isozyme was detected: immunostaining was very intense after IGF‐I treatment and clearly observable after PDGF and EGF stimulation. This result was strongly supported by the in situ confocal microscopy which parallels the Western blot analysis. These data demonstrate that several, but not all, GFs acting through tyrosine kinase receptor induce the intranuclear translocation of PKC α and, because of the dramatic effect of IGF‐I, strengthen the case for a link between the activation of nuclear inositol lipid cycle and PKC translocation induced by this GF.
Existence of nuclear‐encoded 5S‐rRNA in bovine mitochondria
Authors: Yoshionari Shigeo , Koike Tomohiro , Yokogawa Takashi , Nishikawa Kazuya , Ueda Takuya , Miura Kinichiro , Watanabe Kimitsuna
Published: July 2002

A number of proteins functioning in mitochondria are synthesized in the cytoplasm and imported into the mitochondria via specific transport systems. In mammals, on the contrary, mitochondrial membranes have generally been considered to be impermeable to nucleic acids. However, here we show that an RNA with 120 nucleotides, the sequence of which is identical to that of the nuclear‐encoded 5S RNA, exists in bovine mitochondria, although the mitochondrial genome encodes no 5S RNA gene. This RNA molecule was found to be retained in purified bovine mitochondria as well as in the mitoplasts, even after extensive treatment with an RNase, demonstrating that the 5S RNA is actually located inside the mitochondrial inner membrane. The 5S rRNA molecule was also shown to exist in mitochondria from rabbit and chicken.
Molecular mechanisms in the disassembly and reassembly of the mammalian Golgi apparatus during M‐phase
Authors: Misteli Tom
Published: July 2002

The mitotic disassembly and reassembly of the mammalian Golgi apparatus is an iDAal system to study the molecular mechanisms involved in biogenesis and maintenance of membranous organelles. As cells enter M‐phase, Golgi stacks are converted into Golgi clusters of small membrane fragments, which are dispersed throughout the cytoplasmic space during metaphase. Disassembly is DApenDAnt on the action of cdc2‐kinase and at least two distinct pathways contribute to the fragmentation: one involves the budding of COP I‐coated vesicles from Golgi cisternae, the other is a less well characterised COP I‐inDApenDAnt pathway. During telophase, the Golgi fragments reassemble and fuse into a fully functional Golgi stack, using at least two distinct ATPase‐mediated fusion pathways.
Novel substrate specificity of a membrane‐bound β‐glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii
Authors: Matsui Ikuo , Sakai Yukihiro , Matsui Eriko , Kikuchi Hisasi , Kawarabayasi Yutaka , Honda Koichi
Published: July 2002

A β‐glycosidase gene homolog of Pyrococcus horikoshii (BGPh) was successfully expressed in Escherichia coli. The enzyme was localized in a membrane fraction and solubilized with 2.5% Triton X‐100 at 85°C for 15 min. The optimum pH was 6.0 and the optimum temperature was over 100°C, respectively. BGPh stability was dependent on the presence of Triton X‐100, the enzyme's half‐life at 90°C (pH 6.0) was 15 h. BGPh has a novel substrate specificity with k
cat/K
m values high enough for hydrolysis of β‐D‐Glcp derivatives with long alkyl chain at the reducing end and low enough for the hydrolysis of β‐linked glucose dimer more hydrophilic than aryl‐ or alkyl‐β‐D‐Glcp.
Two genes encoding distinct cytosolic glutamine synthetases are closely linked in the pine genome
Authors: Ávila Sáez Concepción , Muñoz-Chapuli Ramón , Plomion Christophe , Frigerio Jean-Marc , Cánovas Francisco M.
Published: July 2002

The major isoenzyme of glutamine synthetase found in leaves of angiosperms is the chloroplastic form. However, pine seedlings contain two cytosolic glutamine synthetases in green cotyledons: GS1a, the predominant isoform, and GS1b, a minor enzyme whose relative amount is increased following phosphinotricin treatment. We have cloned a GS1b cDNA, and comparison with the previously reported GS1a cDNA sequence indicated that they correspond to separate cytosolic GS genes encoding distinct protein products. Phylogenetic analysis showed that the newly reported sequence is closer to cytosolic angiosperm GS than to GS1a, suggesting therefore that GS1a could be a divergent gymnospermous GS1 gene. Gene mapping using a F2 family of maritime pine showed co‐localization of both GS genes on group 2 of the genetic linkage map. This result supports the proposed origin of different members of the GS1 family by adjacent gene duplication. The implications for gymnosperm genome organization are discussed.
Xanthophylls of the major photosynthetic light‐harvesting complex of plants: identification, conformation and dynamics
Authors: Ruban Alexander V. , Pascal Andrew A. , Robert Bruno
Published: July 2002

The electronic transitions of lutein and neoxanthin in the major light‐harvesting complex, LHCIIb, have been identified for the first time. It was found that 0–0, 0–1 and 0–2 transitions of neoxanthin were located around 486, 457 and 430 nm, whilst those for lutein were dependent on the oligomerisation state. For the monomer, the absorption bands of lutein were found at 495, 466 and 437 nm. Trimerisation caused a decrease in lutein absorption and the parallel appearance of an additional absorption band around 510 nm, which was identified by resonance Raman excitation spectra to originate from lutein. Circular dichroism measurements together with analysis of the ν
4 resonance Raman region of xanthophylls suggested that this lutein molecule is distorted in the trimer. This feature is not predicted by the LHCIIb atomic model of Kühlbrandt and co‐workers [Kühlbrandt, W., Wang, D.N. and Fugiyoshi, Y. (1994) Nature 367, 614–621] and is an important step in understanding pigment dynamics of the complex. Oligomerisation of trimers led to a specific distortion of the neoxanthin molecule. These observations suggest that the xanthophylls of LHCIIb sense the protein conformation and which may reflect their special role in the assembly and function of the light‐harvesting antenna of higher plants.
Role of insulin receptor substrate‐2 in interleukin‐9‐dependent proliferation
Authors: Demoulin Jean-Baptiste , Grasso Luigi , Atkins John M. , Stevens Monique , Louahed Jamila , Levitt Roy C. , Nicolaides Nicholas C. , Renauld Jean-Christophe
Published: July 2002

Interleukin‐9 (IL‐9) stimulation results in JAK, STAT and IRS1/2 phosphorylation. The role of IRS adaptor proteins in IL‐9 signaling is not clear. We show that IL‐9 induces IRS2 phosphorylation and association with phosphatidylinositol‐3 kinase (PI 3‐K) p85 subunit in TS1 cells and BaF/9R cells, which proliferate upon IL‐9 stimulation. We observed a PI 3‐K‐dependent phosphorylation of protein kinase B (PKB) in TS1 cells, but not in BaF/9R, nor in other IL‐9‐dependent cell lines. Finally, 32D cells that were transfected with the IL‐9 receptor but lack IRS expression survived in the presence of IL‐9. Ectopic IRS1 expression allowed for IL‐9‐induced proliferation, in the absence of significant PKB phosphorylation.
Rice 1Cys‐peroxiredoxin over‐expressed in transgenic tobacco does not maintain dormancy but enhances antioxidant activity
Authors: Lee Kyun Oh , Jang Ho Hee , Jung Bae Gyo , Chi Yong Hun , Lee Ji Yeun , Choi Yeon Ok , Lee Jung Ro , Lim Chae Oh , Cho Moo Je , Lee Sang Yeol
Published: July 2002

Possible functions that have been proposed for the plant 1Cys‐peroxiredoxin, include activity as a dormancy regulator and as an antioxidant. The transcript level of ice ys‐eroxiedoin (R1C‐Prx) rapidly decreased after imbibition of rice seeds, but the protein was detected for 15 days after imbibition. To investigate the function of this protein, we generated transgenic tobacco plants constitutively expressing the R1C‐Prx gene. The transgenic R1C‐Prx plants showed a germination frequency similar to control plants. However, the transgenic lines exhibited higher resistance against oxidative stress, suggesting that antioxidant activity may be its primary function.
Splice donor site mutation in the lysosomal neuraminidase gene causing exon skipping and complete loss of enzyme activity in a sialidosis patient
Authors: Penzel Roland , Uhl Johannes , Kopitz Jürgen , Beck Michael , Otto Herwart F. , Cantz Michael
Published: July 2002

Sialidosis is a lysosomal storage disease caused by the deficiency of α‐N‐acetylneuraminidase (NEU1; sialidase), the key enzyme for the intralysosomal catabolism of sialylated glycoconjugates. We have identified a homozygous transversion in the last intron (IVSE +1 G>C) in neu1 of a sialidosis patient. Sequencing of the truncated cDNA revealed an alternatively spliced neu1 transcript which lacks the complete sequence of exon 5. Skipping of exon 5 leads to a frameshift and results in a premature termination codon. This is the first description of an intronic point mutation causing a complete deficiency of the lysosomal neuraminidase activity.
Agonist‐independent and ‐dependent oligomerization of dopamine D2 receptors by fusion to fluorescent proteins
Authors: Wurch T , Matsumoto A , Pauwels P.J
Published: July 2002

Oligomerization of the short (D2S) and long (D2L) isoforms of the dopamine D2 receptor was explored in transfected Cos‐7 cells by their C‐terminal fusion to either an enhanced cyan or enhanced yellow fluorescent protein (ECFP or EYFP) and the fluorescent fusion protein interaction was monitored by a fluorescence resonance energy transfer (FRET) assay. The pharmacological properties of the fluorescent fusion proteins, as measured by both displacement of [3H]nemonapride binding and agonist‐mediated stimulation of [35S]GTPγS binding upon co‐expression with a GαoCys351Ile protein, were not different from the respective wild‐type D2S and D2L receptors. Co‐expression of D2S:ECFP+D2S:EYFP in a 1:1 ratio and D2L:ECFP+D2L:EYFP in a 27:1 ratio resulted, respectively, in an increase of 26% and 16% in the EYFP‐specific fluorescent signal. These data are consistent with a close proximity of both D2S and D2L receptor pairs of fluorescent fusion proteins in the absence of ligand. The agonist‐independent D2S receptor oligomerization could be attenuated by co‐expression with either a wild‐type, non‐fluorescent D2S or D2L receptor subtype, but not with a distinct β2‐adrenoceptor. Incubation with the agonist (−)‐norpropylapomorphine dose‐dependently (EC50: 0.23±0.06 nM) increased the FRET signal for the co‐expression of D2S:ECFP and D2S:EYFP, in support of agonist‐dependent D2S receptor oligomerization. In conclusion, our data strongly suggest the occurrence of dopamine D2 receptor oligomers in intact Cos‐7 cells.
The rôle of the proline‐rich region in A1‐type myosin essential light chains: implications for information transmission in the actomyosin complex
Authors: Timson David J , Trayer Ian P
Published: July 2002

The proline‐rich region of A1‐type myosin essential light chains functions as a spacer arm separating an actin binding site at the extreme N‐terminus from the remainder of the protein. Alteration of the length of this region leaving the actin binding site intact results in altered actin‐activated MgATPase kinetics when these light chains are hybridised into myosin subfragment‐1. In the case of a mutant in which the length of the proline‐rich region was doubled, actin binding by the light chain was uncoupled from kinetic modulation. The implications of this result for information transmission in the actomyosin complex are discussed.
Role of carotene in the rapid turnover and assembly of photosystem II in Chlamydomonas reinhardtii
Authors: Trebst Achim , Depka Brigitte
Published: July 2002

Inhibitors of the phytoene desaturase in carotene biosynthesis were tested in the enhanced rapid turnover of the D1 protein of photosystem II in high light exposure of Chlamydomonas reinhardtii cells. After 1 h high light on heterotrophically grown cells in the presence of norflurazon or fluridone, photosynthesis activity in vivo and PS II activity in vitro is lost. The D1 protein has disappeared. PS I activity is not affected, nor is the D2 protein. It is concluded that β‐carotene is essential for the assembly of the D1 protein into functional photosystem II. It is suspected that bleaching of β‐carotene in the reaction center of PS II by high light destabilizes the structure and triggers the degradation of the D1 protein.
Phytoalexin production by amino acid conjugates of jasmonic acid through induction of naringenin‐7‐O‐methyltransferase, a key enzyme on phytoalexin biosynthesis in rice (Oryza sativa L.)
Authors: Tamogami Shigeru , Rakwal Randeep , Kodama Osamu
Published: July 2002

Amino acid conjugates of jasmonic acid are found to elicit production of the flavonoid phytoalexin, sakuranetin in rice leaves. The elicitation is shown to arise from induction of naringenin 7‐O‐methyltransferase, a key enzyme of sakuranetin biosynthesis. The (−)‐phenylalanine conjugate, one of the active compounds, is characterized by high activity for both sakuranetin and enzyme induction and low phytotoxicity against rice growth. Both (+)‐enantiomers of the conjugates and free amino acids do not show any activity. The amino acid conjugate of jasmonic acid is speculated to be the later component in the signaling transduction chain in stressed rice plants.
Restoration of lectin activity to a non‐glycosylated ricin B chain mutant by the introduction of a novel N‐glycosylation site
Authors: Zhan Jinbiao , de Sousa Michelle , Chaddock John A. , Roberts Lynne M. , Lord J.Michael
Published: July 2002

Ricin B chain (RTB) is an N‐glycosylated, galactose‐specific lectin. Removal of the two native N‐glycosylation sites at Asn95 and Asn135 by site‐directed mutagenesis generated a recombinant protein devoid of lectin activity. Two novel N‐glycosylation sites were introduced into RTB at Asn42 and Asn123, either singly or in combination. Microinjection of pre‐RTB transcripts into Xenopus oocytes showed that these novel sites became glycosylated in vivo. The single oligosaccharide site chain at Asn42 restored lectin activity to RTB, whereas glycosylation at Asn123 or simultaneous glycosylation at Asn42 and Asn123 failed to do so.
The G protein α subunit (GPα1) is associated with the ER and the plasma membrane in meristematic cells of Arabidopsis and cauliflower
Authors: Weiss Catherine A , White Elyssa , Huang Hai , Ma Hong
Published: July 2002

Towards the elucidation of the cellular function(s) of GPα1, we have characterized its subcellular localization using immunofluorescence and cell fractionation. GPα1 is not present in nuclei or chloroplasts. It is a membrane‐bound protein, and analysis of isolated endoplasmic and plasma membranes indicates a good correlation between GPα1 in both the plasma membrane and the ER compartment. Interestingly, these results may suggest more different functions for GPα1: it might be involved in transmission of extracellular signals across the plasma membrane and in the cytoplasm, and/or it may also be involved in regulating some aspects of the ER functions or membrane trafficking between both membranes.
Disulfide‐bonding between Drosophila laminin β and γ chains is essential for α chain to form αβγ trimer
Authors: Kumagai Chino , Kadowaki Tatsuhiko , Kitagawa Yasuo
Published: July 2002

Assembly of Drosophila laminin α, β and γ chains was analyzed by immunoprecipitation of the lysate from metabolically radiolabeled Kc 167 cells with chain‐specific antibodies followed by two dimensional electrophoresis in which non‐reducing and reducing SDS gel electrophoresis are combined. Precipitation of monomeric β (or γ) with anti‐γ (or ‐β) antibody revealed that β and γ form stable dimer before they are disulfide‐bonded to each other. In contrast, α associates with neither monomeric β, monomeric γ nor βγ dimer without disulfide‐bonding but only with disulfide‐bonded βγ dimer to form αβγ trimers. These results thus demonstrated that the interchain disulfide‐boding between β and γ is essential for α to form αβγ trimer. We also found that the αβγ trimer can be secreted with α chain either disulfide‐bonded or not bonded to the disulfide‐bonded βγ dimer.
Limited photosynthetic electron flow but no CO2 fixation in Chlamydomonas mutants lacking photosystem I
Authors: Cournac Laurent , Redding Kevin , Bennoun Pierre , Peltier Gilles
Published: July 2002

By measuring O2 and CO2 exchange in mutants of the green alga Chlamydomonas reinhardtii in which genes encoding the reaction center of photosystem I (psaA or psaB) have been deleted, we found that a photosystem II‐dependent electron flow using O2 as the final acceptor can be sustained in the light. However, in contrast with recent reports using other Chlamydomonas mutants (B4 and F8), we show here that CO2 fixation does not occur in the absence of photosystem I. By deleting the psaA gene in both B4 and F8 strains, we conclude that the ability of these mutants to fix CO2 in the light is due to the presence of residual amounts of photosystem I.
Acid induced equilibrium unfolding of annexin V wild type shows two intermediate states
Authors: Beermann Br.Bernd , Hinz Hans-Jürgen , Hofmann Andreas , Huber Robert
Published: July 2002

Annexin V is an α‐helical protein which shows anticoagulatory and antiinflammatory activity. It is supposed to be involved in membrane fusion and exocytosis. In this study acid‐induced equilibrium unfolding of the human annexin V is investigated by fluorescence and circular dichroism spectroscopy. The spectroscopic data indicate that at least two intermediate states are involved in unfolding. One of the proposed intermediate states exhibits properties similar to those observed with annexin V wild type saturated with calcium, another may be regarded as ‘molten globule’.
Elucidation of the cause for reduced activity of abnormal human plasmin containing an Ala55‐Thr mutation: importance of highly conserved Ala55 in serine proteases
Authors: Takeda-Shitaka Mayuko , Umeyama Hideaki
Published: July 2002

In serine proteases, Ala55 is highly conserved and located just behind the catalytic triad. That the activity of human plasmin is reduced by the A55T substitution indicates the importance of Ala55 in catalysis. In the present study, the 3‐D model of A55T human plasmin shows that an unusual hydrogen bond between Thr55 Oγ1 and His57 Nϵ2 alters His57 into an inactive conformation in which His57 cannot accept a proton from Ser195 as a catalytic base. Our results demonstrate that Ala55 contributes heavily to the active conformation of His57 and ensures the proton transfer from Ser195 to His57.
Indirect evidence for structural changes coupled with Q−⋅B formation in photosystem II
Authors: Reifarth F , Renger G
Published: July 2002

The thermal blockage of Q−⋅
A oxidation was analysed in PS II membrane fragments by monitoring flash‐induced changes of the relative fluorescence quantum yield as a function of temperature. The results obtained reveal: (a) in dark‐adapted samples the fraction of Q−⋅
A that is not reoxidised within a time domain of 10 s after the actinic flash increases with lowering the temperature (half‐maximum effect at 250–260 K), (b) at low temperatures where Q−⋅
A generated in dark‐adapted samples remains almost completely reduced, a significant extent of Q−⋅
A reoxidation arises when samples are used that were preilluminated at room temperature by one saturating flash followed by rapid freezing before performing the experiment, and (c) the extent of Q−⋅
A that is reoxidised at 258 K exhibits a characteristic binary oscillation as a function of the number of preillumination flashes given at room temperature. Based on these data it is inferred that QB and Q−⋅
B are located at different equilibrium positions in the QB site. As a consequence the formation of Q−⋅
B is coupled with significant structural changes that require sufficient flexibility of the protein matrix. This general feature corresponds with a recently proposed model for the acceptor side reactions of anoxygenic bacteria [Stowell, M.H.B., McPhillips, T.M., Rees, D.C., Soltis, S.M., Abresch, E. and Feher, G., Science 276 (1997) 812–816].
Intestinal lactase‐phlorizin hydrolase (LPH): the two catalytic sites; the role of the pancreas in pro‐LPH maturation
Authors: Zecca Laura , Mesonero José E , Stutz Alfred , Poirée Jean-Claude , Giudicelli Jean , Cursio Raffaele , Gloor Sergio M , Semenza Giorgio
Published: July 2002

The proteolytic processing of pro‐lactase‐phlorizin hydrolase by (rat) enterocytes stops two amino acid residues short of the N‐terminus of ‘mature’ final, brush border lactase‐phlorizin hydrolase. Only these two amino acid residues are removed by luminal pancreatic protease(s), probably trypsin.
Reactions of denatured proteins with other cellular components to form insoluble aggregates and protection by lactoferrin
Authors: Takase Kenji
Published: July 2002

Elucidation of the mechanism of formation of insoluble protein aggregates is essential to resolve problems such as protein folding diseases. In this study the effects of various types of biomolecules on the aggregation of denatured proteins were investigated. Denatured α‐lactalbumin, an acidic protein, was found to be precipitated by lactoferricin, a basic peptide derived from lactoferrin. Denatured lysozyme, a basic protein, by itself showed aggregation, which was promoted by addition of native α‐lactalbumin. Heparin and nucleic acids caused almost instant aggregation of denatured lysozyme. Native lactoferricin was also found to aggregate with heparin or nucleic acids. The results show that denatured/misfolded proteins as well as peptides are highly reactive with other cellular components to form insoluble aggregates and suggest a possible mechanism by which protein folding diseases progress. Most of the above aggregation reactions were inhibited by lactoferrin, which could form soluble complexes with denatured α‐lactalbumin, heparin, and nucleic acids.