bluuebunny/crossref_metadata_2025 huggingface dataset:
A critical examination of the role of de novo protein synthesis in the osmotic adaptation of the halotolerant alga Dunaliella
Authors: Sadka Avi , Lers Amnon , Zamir Ada , Avron Mordhay
Published: July 2002

The role of protein synthesis during the osmotic adaptation of Dunaliella salina was examined utilizing protein synthesis inhibitors. 3–4 h of preincubation with cycloheximide or chloramphenicol were required to observe full inhibition of protein synthesis. Algae treated with cycloheximide, chloramphenicol or both reagents prior to a hyper‐ or hypo‐osmotic shock showed no significant difference in their osmotic response when compared to untreated controls. Thus, protein synthesis is not essential for the osmotic adaptation response. Cell division was halted following a hyper—osmotic shock, but proliferation was reinitiated already during the late phase of the osmotic response and exponential growth was resumed soon after completion of the osmotic response.
ATP synthesis coupled to electron transfer from H2 to the heterodisulfide of 2‐mercaptoethanesulfonate and 7‐mercaptoheptanoylthreonine phosphate in vesicle preparations of the methanogenic bacterium strain Gö1
Authors: Peinemann S. , Hedderich R. , Blaut M. , Thauer R.K. , Gottschalk G.
Published: July 2002

Crude vesicle preparations of the methanogenic strain Gol were able to couple the reduction of the heterodisulfide of 2‐mercaptoethanesulfonate and 7‐mercaptoheptanoylthreonine phosphate (CoM‐S‐S‐HTP) by H2 with ATP formation. The rate of ATP synthesis was 1 mg protein. ATP synthesis and disulfide reduction were only observed with CoM‐S‐S‐HTP, but not with CoM‐S‐S‐CoM or HTP‐S‐S‐HTP. The methylreductase inhibitor 2‐bromoethanesulfonic acid had no effect on ATP synthesis induced by CoM‐S‐S‐HTP reduction with H2. ATP synthesis was completely inhibited by the uncoupler SF 6847 whereas the concomitant CoM‐S‐S‐HTP reduction was stimulated. The ATP synthase inhibitors DCCD and DES also inhibited ATP formation completely and decreased the CoM‐S‐S‐HTP reduction rate to 35% of the control. These inhibitory effects were abolished by addition of the uncoupler. From these results it is concluded that energy coupling between the electron transfer from H2 to the heterodisulfide and ATP synthesis occurs via a transmembrane proton gradient.
Mapping of the region of the tick‐borne encephalitis virus replicase adjacent to initiating substrate binding center
Authors: Morozova O.V. , Mustaev A.A. , Belyavskaya N.A. , Zaychikov E.F. , Kvetkova E.A. , Wolf Yu.I. , Pletnev A.G.
Published: July 2002

Affinity labelling with aldehyde‐containing analogs of initiation substrates of nuclear fraction of tick‐borne encephalitis virus (TBEV) infected cells results in a labelling of a single polypeptide with a molecular mass of 68 kDa which was immunologically identified as TBEV NS3 protein. A single‐hit hydroxylamine hydrolysis, using limited and long‐term CNBr cleavages allowed one to identify Lys1800 and/or Lys1803 as the label attachment sites. These amino acid residues are situated in the proximity of the ‘B’‐site of NTP‐binding motif of viral RNA replicase.
D1‐D2 complex of the photosystem II reaction center from spinach Isolation and partial characterization
Authors: Tang Xiao-Song , Fushimi Kazuo , Satoh Kimiyuki
Published: July 2002

A pigment‐protein complex consisting of D1 and D2 proteins, but depleted in the two lower molecular mass components of photosystem II, i.e. cytochrome b‐559 and psbI gene product, has been isolated by octyl‐β‐D‐glucopyranoside treatment of the purified photosystem II reaction center complex from spinach [(1987) Proc. Natl. Acad. Sci. USA 84, 109‐112], followed by separation by high performance liquid chromatography using a gel‐permeation column (TSK G3000 SW). The isolated complex is photochemically active in the photoreduction of intrinsic pheophytin a under steady‐state illumination, in the presence of dithionite and methyl viologen, and exhibits pigment stoichiometries similar to those in the untreated reaction center, indicating that the D1‐D2 complex provides the site of primary photochemistry in photosystem II, as well as the principal binding sites of pigments in the reaction center.
Clear cell carcinoma of the human ovary synthesizes and secretes a transferrin with microheterogeneity of lectin affinity
Authors: Ohkawa Kiyoshi , Takada Koji , Takizawa Naoko , Hatano Takashi , Tsukada Yutaka , Matsuda Makoto
Published: July 2002

Human ovarian clear cell carcinoma cell line (transferrin (Tf)‐non‐producer), HAC 2, cells were adapted to grow in chemically defined synthetic medium when the cells were cultured with medium containing 10 of insulin at least for 6 months. They synthesized and secreted constantly the 80 kDa protein immunologically similar to human serum Tf(15 ± 12 /107 cells/3 days). By sensitive lectin‐affinity electrophoresis followed by antibody‐affinity blotting technique, a concanavalin A weakly bound or unbound, lentil lectin, a strongly reactive abnormal band, which was rarely found in human serum Tf, was detectable in the Tf synthesized by HAC 2 cells (HACTf). These findings suggest that the HACTf may act as one of the autocrine growth factors and that this heterogeneity of HACTf for lectin affinity is ascribed to differences in the carbohydrate moiety of the Tf.
Adherence of human neutrophils changes Ca2+ signaling during activation with opsonized particles
Authors: Koenderman Leo , Tool Anton T.J. , HooyBrink Berend , Roos Dirk , Hansen Carl A. , Williamson John R. , Verhoeven Arthur J.
Published: July 2002

Changes in the cytosolic free Ca2+ concentration ([Ca2+]i) upon activation of human neutrophils by opsonized particles (serum‐treated zymosan; STZ) were evaluated by three different methods: (i) measurement of total fluorescence changes in indo‐1 loaded neutrophils activated in suspension; (ii) measurement of fluorescence changes in individual indo‐1 loaded neutrophils in a flow cytometer and (iii) measurement of fluorescence changes in individual fura‐2 loaded neutrophils adherent to serum‐coated coverslips. Our study shows that the opsonized particle‐induced change in [Ca2+]i in neutrophils is altered during adherence of the cells to a serum‐coated surface. These observations might be of importance for neutrophil function in vivo, since adherence is a prerequisite for diapedesis and chemotaxis.
Botulinum ADP‐ribosyltransferase activity as affected by detergents and phospholipids
Authors: Maehama Tomohiko , Ohoka Yoshiharu , Ohtsuka Tomokazu , Takahashi Katsunobu , Nagata Koh-ichi , Nozawa Yoshinori , Ueno Kazue , Ui Michio , Katada Toshiaki
Published: July 2002

GTP‐binding proteins with Mr
values of 22 000 and 25 000 in bovine brain cytosol were ADP‐ribosylated by an exoenzyme (termed C3) purified from Clostridium botulinum type C. The rate of C3‐catalyzed ADP‐ribosylation of the partially purified substrates was extremely low by itself, but was increased enormously when a protein factor(s) obtained from the cytosol was simultaneously added. The rate of the C3‐catalyzed reaction was also stimulated by the addition of certain types of detergents or phospholipids even in the absence of the protein factors. The ADP‐ribosylation appeared to be enhanced to an extent more than the additive effect of either the protein factors or the detergents (and phospholipids). Thus, ADP‐ribosylation catalyzed by botulinum C3 enzyme was affected not only by cytoplasmic protein factors but also by detergents or phospholipids in manners different from each other.
The protecting effect of L‐carnitine on Ca2+‐loaded rat liver mitochondria
Authors: Mokhova E. , Arrigoni-Martelli E. , Bellei M. , Dedukhova V. , Muscatello U. , Starkov A. , Bobyleva V.
Published: July 2002

It is shown that L‐carnitine strongly increases the ability of rat liver mitochondria to respond to the train of Ca2+ additions by a transient stimulation of the State‐4 respiration rate. Such an effect requires ATP and the L‐carnitine efficiency strongly decreases when ATP is omitted, Oleate influences the mitochondria in a fashion opposite to that of L‐carnitine. The oleate effect is strongly diminished by L‐carnitine. Again, the L‐carnitine effect requires ATP, and D‐carnitine fails to substitute for L‐carnitine. It is suggested that L‐carnitine removes, in an ATP‐dependent manner, endogenous or added fatty acids, which are involved in oxidative damage of Ca2+‐loaded mitochondria.
Protein phosphorylation stimulates the rate of malate uptake across the peribacteroid membrane of soybean nodules
Authors: Ouyang Li-Jun , Whelan James , Weaver C.David , Roberts Daniel M. , Day David A.
Published: July 2002

It is suggested that the rate of malate uptake across the peribacteroid membrane is controlled by phosphorylation of nodulin 26.
Functional expression of N‐terminal truncated α‐subunits of Na,K‐ATPase in Xenopus laevis oocytes
Authors: Burgener-Kairuz Pauline , Horisberger Jean-Daniel , Geering Käthi , Rossier Bernard C.
Published: July 2002

N‐terminal deletion mutants of Na,K‐ATPase α1 isoforms initiating translation at Met34 (α1T1) or at Met43 (α1T2) were expressed in X. laevis oocytes. Compared to β3 cRNA injected controls, the co‐expression of α1wt, α1T1, α1T2 with β3 subunits results in a 2‐ to 3‐fold increase of ouabain binding sites, parallelled by a concomitant increase in Na,K‐pump current. The apparent K½ for potassium activation of the α1T2/β3 Na,K‐pumps is significantly higher than that of the α1wt/β3 or α1T1/β3 Na,K‐pumps expressed at the cell surface. Total deletion of the lysine‐rich N‐terminal domain thus allows the expression of active Na,K‐pump but with distinct cation transport properties.
Pseudomonas aeruginosa acid phosphatase Activation by divalent cations and inhibition by aluminium ion
Authors: Domenech Carlos E. , Lisa Teresita A. , Salvano Mario A. , Garrido Mónica N.
Published: July 2002

In Pseudomonas aeruginosa, the effect of different cations on the acid phosphatase activity was studied in order to acquire more information related to a previously proposed mechanism, involving the coordinated action of this enzyme with phospholipase C. Although the natural substrate of this enzyme is phosphorylcholine, in order to avoid the possible interaction of its positive charge and those of the different cations with the enzyme molecule, the artificial substrate p‐nitrophenylphosphate was utilized. Kinetic studies of the activation of acid phosphatase (phosphorycholine phosphatase) mediated by divalent cations Mg2+, Zn2+ and Cu2+ revealed that all these ions bind to the enzyme in a compulsory order (ordered bireactant system). The Km
values obtained for p‐NPP in the presence of Mg2+, Zn2+ and Cu2+ were 1.4 mM, 1.0 mM and 3.5 mM, respectively. The K
A values for the same ions were 1.25 mM, 0.05 mM and 0.03 mM, respectively. The V
max obtained in the presence of Cu2+ was about twofold higher than that obtained in the presence of Mg2+ or Zn2+. The inhibition observed with Al3+ seems to be a multi‐site inhibition, The K′app and n values, from the Hill plot, were about 0.25 mM and 4.0 mM, respectively, which were independent of the metal ion utilized as activator. It is proposed that the acid phosphatase may excert its action under physiological conditions, depending on the availability of either one of these metal ions.
A novel 15N‐labeling method to selectively observe 15NH2 resonances of proteins in 1H‐detected heteronuclear correlation spectroscopy
Authors: Tate Shin-ichi , Tate Naoko U. , Ravera Mark W. , Jaye Michael , Inagaki Fuyuhiko
Published: July 2002

An experimental method to selectively label side‐chain NH2 groups of glutamine and asparagine in proteins with 15N is proposed. This selective labeling method enables to observe only 15NH2 resonances and thus, to discriminate between 15NH and 15NH2 resonances in a 1H‐detected heteronuclear correlation spectrum. This method gives results with approximately two times higher sensitivity than those obtained by elaborate pulse sequences such as DEPT‐HSQC and will be useful for studying the molecular interaction involving the side chains of Asn and Gln residues.
On the catalytic mechanism of EcoRI and EcoRV A detailed proposal based on biochemical results, structural data and molecular modelling
Authors: Jeltsch Albert , Alves Jürgen , Maass Günter , Pingoud Alfred
Published: July 2002

EcoRI and EcoRV have a very similar active site, as is apparent from a comparison of the structures of their respective protein‐DNA complexes. Based on structural and mechanistic data, as well as detailed molecular modelling presented here, a mechanism for the DNA cleavage by these enzymes is suggested in which the attacking water molecule is activated by the phosphate group 3′ to the scissile phosphodiester bond, and in which the leaving group is protonated by a water molecule associated with the essential cofactor, Mg2+. The mechanism proposed may also apply to other nucleases.
The primitive metazoan Hydra expresses antistasin, a serine protease inhibitor of vertebrate blood coagulation: cDNA cloning, cellular localisation and developmental regulation
Authors: Holstein Thomas W. , Mala Carola , Kurz Eva , Bauer Karin , Greber Martin , David Charles N.
Published: July 2002

We have isolated and characterized cDNAs from Hydra which encode antistasin, a potent inhibitor of factor Xa in the vertebrate blood clotting cascade. Hydra antistasin is expressed in gland cells and represents a major class of transcripts from Hydra's head. Sequence analysis revealed that Hydra antistasin contains 6 internal repeats of a 25–26 amino acid sequence with a highly conserved pattern of 6 cysteine and 2 glycine residues identical to that in leech antistasin. Conservation of antistasin in a lower metazoan provides a potential link between the vertebrate and invertebrate coagulation systems.
Photoinhibition affects the non‐heme iron center in photosystem II
Authors: Gleiter Hermann M. , Nugent Jonathan H.A. , Haag Elisabeth , Renger Gernot
Published: July 2002

Effects on the PS II acceptor side caused by exposure to strong white light (180 W/m2) of PS II membrane fragments (spinach) at pH 6.5 and 0°C were analyzed by measuring low temperature EPR signals and flash‐induced transient changes of the flourescence quantum yield. The following results were obtained: (a) the extent of the light induced g = 1.9 EPR signal as a measure of photochemical Fe2+Q−
A formation declines with progressing photoinhibition. The half‐life of this effect is independent of the absence or presence of an exogenous electron acceptor during the photoinhibitory treatment; (b) in samples photoinhibited in the absence of an electron acceptor and subsequently incubated with K3[Fe(CN)6] in the dark, the extent of the g = 8 EPR signal (reflecting the oxidized Fe3+ form of the endogenous non‐heme iron center) and of the flash‐induced change of the flourescence yield (as a measure of fast electron transfer from Q−
A to Fe3+ after the first flash; [see (1992) Photosynth. Res. 31, 113–126] exhibits the same dependence on photoinhibition time as the g = 1.9 EPR signal; (c) in samples photoinhibited in the presence of an exogenous electron acceptor, the signals reflecting Fe3+‐formation and fast electron transfer from Q−
A to Fe3+ decline faster than the g = 1.9 EPR signal. These results provide for the first time direct evidence that the endogenous non‐heme iron located between QA and QB is susceptible to modifications by light stress. The implications of this finding will be discussed.
Essential catalytic role of Glu134 in endo‐β‐1,3‐1,4‐d‐glucan 4‐glucanohydrolase from B. licheniformis as determined by site‐directed mutagenesis
Authors: Planas A. , Juncosa M. , Lloberas J. , Querol E.
Published: July 2002

Site‐directed mutagenesis experiments designed to identify the active site of Bacillus licheniformis endo‐β‐1,3‐1,4‐d‐glucan 4‐glucanohydrolase (β‐glucanase) have been performed. Putative catalytic residues were chosen on the basis of sequence similarity analysis to viral and eukaryotic lysozymes. Four mutant enzymes were expressed and purified from recombinan: E. coli and their kinetics analysed with barley β‐glucan. Replacement of Glu134 by Gin produced a mutant (E134Q) that retains less than 0.3% of the wild‐type activity. The other mutants, D133N, E160Q and D179N, are active but show different kinetic parameters relative to wild‐type indicative of their participation in substrate binding and transition‐state complex stabilization. Glu134 is essential for activity; it is comprised in a region of high sequence similarity to the active site of T4 lysozyme and matches the position of the general acid catalyst. These results strongly support a lysozyme‐like mechanism for this family of Bacillus β‐glucan hydrolases with Glu134 being the essential acid catalyst.
Primary structure and functional expression of a cyclic nucleotidegated channel from rabbit aorta
Authors: Biel Martin , Altenhofen Wolfram , Hullin Roger , Ludwig Jost , Freichel Marc , Flockerzi Veit , Dascal Nathan , Kaupp U.Benjamin , Hofmann Franz
Published: July 2002

Sequences specific for cyclic nucleotide‐gated channels (CNG channels) have been amplified by PCR from cDNA of heart, aorta, sinoatrial node, cerebellum, C‐cells and kidney. The complete amino acid sequence of a CNG channel from rabbit aorta has been deduced by cloning and sequence analysis of the cDNA. Synthetic RNA derived from this cDNA induces the formation of a functional CNG channel in Xenopus oocytes.
Identification of the barstar binding site of barnase by NMR spectroscopy and hydrogen‐deuterium exchange
Authors: Jones David N.M. , Bycroft Mark , Lubienski Michael J. , Fersht Alan R.
Published: July 2002

The extracellular ribonuclease from Bacillus amyloliquifaciens, barnase, forms a tightly‐bound one‐to‐one complex with its intracellular inhibitor barstar. The barstar binding site on barnase was characterised by comparing the differences in the chemical shift and hydrogen‐deuterium exchange rates between free and bound barnase. Chemical shift assignments of barnase in the complex with barstar were determined from 3D NOESY‐HMQC and TOCSY‐HMQC spectra of a complex that had been prepared with uniformly 15N‐labelled barnase and unlabelled barstar. Hydrogen exchange rates were obtained from an analysis of a series of [15N]HMQC spectra of a sample prepared in the same manner exchanged into D2O. The largest changes in either chemical shift or hydrogen‐deuterium exchange rate are observed for residues located in the active‐site and substrate binding loops indicating that barstar inhibits barnase activity by sterically blocking the active site.
Identification of the psbH gene product as a 6 kDa phosphoprotein in the cyanobacterium Synechocystis 6803
Authors: Race Helen L. , Gounaris Kleoniki
Published: July 2002

The product of the psbH gene has been identified in Synechocystis 6803 thylakoid membranes as a 6 kDa phosphoprotein. This protein becomes phosphorylated in vitro despite the fact that in cyanobacteria it is truncated at the N‐terminus such that the phosphorylation site identified in the higher plant protein is missing. Phosphorylation occurred both in the light and in the dark but was inhibited by oxidising conditions, DCMU and zinc ions. The cyanobacterial 6 kDa phosphoprotein degrades when the membranes are subjected to high intensity illumination.
Glyceraldehyde‐3‐phosphate activates auto‐ADP‐ribosylation of glyceraldehyde‐3‐phosphate dehydrogenase
Authors: Kots Alexander Ya. , Sergienko Edward A. , Bulargina Tamara V. , Severin Eugene S.
Published: July 2002

Nitric oxide was recently demonstrated to stimulate ADP‐ribosylation of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). Our studies on the effect of glyceraldehyde‐3‐phosphate (GA3P), the natural substrate of dehydrogenase activity of GAPDH, indicated GA3P to be another very potent activator of ADP‐ribosylation of the enzyme. GA3P was able to activate ADP‐ribosylation only in the presence of DTT. The action of GA3P was associated with inhibition of GAPDH dehydrogenase activity. K
a for GA3P was at least 50‐fold lower and maximal activation was somewhat higher than these values for other aldehydes that were also able to enhance GAPDH ADP‐ribosylation in the presence of DTT. ADP‐ribosylation was blocked by carboxamidomethylation of the essential cysteine SH‐group. The bond between the prelabeled protein and ADP‐ribose was resistant to hydrolysis with hydroxylamine and HgCl2, suggesting that a lysine ε‐amino group is the target for ADP‐ribosylation.
The structure of human carbonic anhydrase II in complex with bromide and azide
Authors: Jönsson B.M. , Håkansson K. , Liljas A.
Published: July 2002

The three‐dimensional structure of human carbonic anhydrase II complexed with azide and with bromide was investigated crystallographically. Both of these non‐protonated inhibitors replace the zinc and the ‘deep’ water, two catalytically important water molecules in the active site of the molecule. Both the azide and the bromide ions bind in a distorted tetrahedral manner 0.4 and 1.1 Å from the zinc water position, respectively, but are in close contact (2.0 and 2.6 Å, respectively) with the zinc ion.
Relative stability of AT and GC pairs in parallel DNA duplex formed by a natural sequence
Authors: Borisova O.F. , Shchyolkina A.K. , Chernov B.K. , Tchurikov N.A.
Published: July 2002

The low‐cooperative melting of parallel DNA formed by a natural 40 bp long sequence from Drosophila: 5′‐d(TGATTGATCGATTGTTTGCATGCACACGTTTTTGTGAGCG)‐3′ 5′‐d(ACTAACTAGCTAACAAACGTACGTGTGCAAAAACACTCGC)‐3′ that possesses a normal nucleotide content was studied by using the special method of measuring the fluorescence of its complex with acriflavine as well as by conventional thermal denaturation. Acriflavine allows discrimination of the melting of AT and GC pairs because its fluorescence is quenched by neighbouring G bases. We have observed that about 40% of AT pairs melt at 14°C while the remainder melt at 42°C. The GC pairs remain stable up to ∼ 40°C and melt at 54°C. The higher stability of GC pairs suggests the formation of cis Watson‐Crick pairs in parallel DNA.
The oxidation of α‐tocopherol in human low‐density lipoprotein by the simultaneous generation of superoxide and nitric oxide
Authors: Hogg Neil , Darley-Usmar Victor M. , Wilson Michael T. , Moncada Salvador
Published: July 2002

Peroxynitrite is the product of the reaction between nitric oxide and superoxide. It is an oxidant which can also decompose to form the hydroxyl radical and nitrogen dioxide. In this report we show that a powerful oxidant with reactivity similar to that of the hydroxyl radical is formed from the generation of superoxide from xanthine oxidase and nitric oxide from S‐nitroso‐n‐acetylpenicillamine (SNAP). Simultaneous generation of these two radicals by either xanthine oxidase/SNAP or the sydnonimine SIN‐1 in the presence of low‐density lipoprotein (LDL) results in the depletion of α‐tocopherol and formation of its oxidised product α‐tocopheroquinone. The mechanism of oxidation required both the formation of nitric oxide and superoxide. In contrast to the promotion of LDL oxidation by transition metals the oxidation of LDL by SIN‐1 was not sensitive to the addition of exogenous lipid hydroperoxide.
Peroxynitrite causes calcium efflux from mitochondria which is prevented by Cyclosporin A
Authors: Packer Michael A. , Murphy Michael P.
Published: July 2002

Superoxide reacts with nitric oxide to form peroxynitrite, a potent oxidising agent which may contribute to tissue damage in pathological situations such as inflammation and ischaemia/reperfusion. One mechanism by which oxidative stress damages tissues is the induction of a specific Cyclosporin A‐sensitive mitochondrial calcium efflux pathway. Here we show that peroxynitrite induces calcium efflux from mammalian mitochondria and that this efflux is blocked by Cyclosporin A. These data suggest that disruption of mitochondrial calcium efflux may contribute to tissue damage when superoxide and nitric oxide are present together in vivo.
Cloning and characterization of the human V3 pituitary vasopressin receptor
Authors: de Keyzer Yves , Auzan Colette , Lenne Frédéric , Beldjord Chérif , Thibonnier Marc , Bertagna Xavier , Clauser Eric
Published: July 2002

Arginine‐vasopressin (AVP) plays a determinant role in the normal ACTH response to stress in mammals. We cloned a human cDNA coding a 424 amino acid G‐protein coupled receptor structurally related to the vasopressin/oxytocin receptor family. When expressed in COS cells, this receptor binds AVP with a high affinity (K
d = 0.55 ± 0.13 nM) and is functionally coupled to phospholipase C. Competition studies with peptidic or non peptidic AVP analogues reveal that it is pharmacologically distinct from V1a and V2 AVP receptors and therefore it is designated V3. RT‐PCR analysis shows that the human V3 receptor is expressed in normal pituitary and also in kidney, but is undetectable in liver, myometrium and adrenal gland. Northern blot analysis reveals a ∼4.8 kb messenger in human corticotropic pituitary adenomas.
Spectral and thermodynamic properties of the two hemes of the D1D2cytochrome b‐559 complex of spinach
Authors: Shuvalov V.A. , Schreiber U. , Heber U.
Published: July 2002

In agreement with previous work [Shuvalov, Heber and Schreiber (1988) FEBS Lett. 258, 27‐31] two hemes (low potential (LP) and extra low potential (XLP )) per two pheophytins were found in isolated D1D2Cyt b‐559 complexes. Reductive and oxidative redox titrations demonstrate that the E
m of the LP form is at about +150 mV. It is independent of pH between pH 7.2 and 9.4. The XLP heme is autoxidizable at pH 7.2 and displays, at this pH, an E
m of − 45 mV. Both the LP and XLP hemes show absorption peaks at 559 nm. They are proposed to have bis‐histidine ligation of the heme iron. At pH 9.4, the XLP heme splits into two forms. One of them has an E
m of +40 mV, and absorption peaks at 559 nm showing the bis‐histidine ligation. The other displays an E
m of −220 mV and the peak is shifted to 562 nm. This last form is proposed to be due to the incorporation of OH− which occupies the 6th coordination position of the heme Fe(III) at high pH. The pK value for the conversion of the XLP heme is close to 7.7. In a structure simulation of the α‐helices of α‐ and β‐polypeptides, the β‐polypeptide, but not the α‐polypeptide, reveals a distance between the histidine N and the heme Fe which permits stable N‐Fe coordination. In the α‐polypeptide, OH− can be incorporated between N and Fe. The functional role of the two hemes of cyt b‐559 is briefly discussed with respect to water oxidation and cyclic electron transfer.
Two hypotheses ‐ one answer
Authors: Soppa Jörg
Published: July 2002

The structure of bacteriorhodopsin (BR) of Halobacterium halobium is known. Despite the lack of sequence similarities it is often taken as a model for eukaryotic G‐protein‐coupled receptors (GPCRs). Recently two hypotheses were used to support the homology of BR and GPCRs, namly evolution by exon shuffling and evolution by gene duplication. BR is a member of a family of halobacterial retinal proteins. The sequences of eight members of this family were used to test the two hypotheses. Based on sequence comparison, no indication for an evolutionary linkage between the two protein families could be found.
Conversion of the sodium channel activator aconitine into a potent α7‐selective nicotinic ligand
Authors: Hardick David J. , Cooper Gary , Scott-Ward Toby , Blagbrough Ian S. , Potter Barry V.L. , Wonnacott Susan
Published: July 2002

Methyllycaconitine (MLA) is a competitive antagonist of nicotinic acetylcholine receptors, with a remarkable preference for neuronal [125I]αBgt binding sites. We have begun to investigate the structural basis of its potency and subtype selectivity. MLA is a substituted norditerpenoid alkaloid linked to a 2‐(methylsuccinimido)benzoyl moiety. Hydrolysis of the ester bond in MLA to produce lycoctonine diminished affinity for rat brain [125I]αBgt binding sites 2500‐fold and abolished affinity for [3H]nicotine and muscle [125I]αBgt binding sites. The voltage‐gated Na+ channel activator aconitine, also a norditerpenoid alkaloid, but with significant structural differences from lycoctonine, displayed comparable weak or absent nicotinic activity. Addition of a 2‐(methylsuccinimido)benzoyl sidechain to O‐demethylated aconitine, to mimic MLA, abolished Na+ channel activation and conferred nanomolar affinity for brain [125I]αBgt binding sites, comparable to that of MLA. We propose that the ester‐linked 2‐(methylsuccinimido)benzoyl group is necessary for nicotinic potency, but α7 selectivity resides in the norditerpenoid core of the molecule.
The purification of ammonia monooxygenase from Paracoccus denitrficans
Authors: Moir James W.B. , Crossman Lisa C. , Spiro Stephen , Richardson David J.
Published: July 2002

The heterotrophic nitrifier Paracoccus denitrificans expresses a membrane‐associated ammonia monooxygenase. The active enzyme has been solubilized in the detergent dodecyl‐β‐d‐maltoside and purified by standard chromatographic techniques. This is the first purification of an ammonia monooxygenase. The enzyme consists of two subunits with molecular masses of 38 and 46 kDa. The purified enzyme is a quinol oxidase, is inhibited by light and a variety of chelating agents and is activated by cupric ions. These properties indicate that this enzyme has similarities to a family of enzymes including the ammonia monooxygenase from Nitrosomonas europaea and the particulate methane monooxygenase from Methylococcus capsulatus (Bath).
Blood coagulation changes in pregnant women at risk of developing preeclampsia
Authors: Savelieva G.M. , Efimov V.S. , Grishin V.L. , Shalina R.I. , Kashezheva A.Z.
Published: July 2002

Objectives: A group of 50 pregnant patients were studied with regard to the time of onset of hypercoagulation, its character and mechanisms of initiation in the development of preeclampsia. Methods: The blood coagulation characteristics, antithrombin (AT)‐III and endogenous heparin levels were studied. All of the patients were designated at risk of developing preeclampsia at the time of registration at the antenatal clinic. Results: Retrospective evaluation shows that endogenous heparin levels and AT‐III activity decreased by nearly 50% within 15 days of the development of preeclampsia and were particularly low 1–7 days before the onset of clinical signs of preeclampsia. Conclusions: These findings indicate that abnormalities in AT activity are a marker of hypercoagulation and begin more than 2 weeks before the development of clinical signs of preeclampsia.
Schizophrenia associated with Klippel-Feil syndrome and flat foot
Authors: Varma SL , Dharap AS , Kareem BA , Aye A , Azhar MZ
Published: July 2002

A 20 year-old patient presented with schizophrenia which was associated with Klippel-Feil syndrome and flat foot.
Prediction of the tertiary structure of a caspase‐9/inhibitor complex
Authors: Chou Kuo-Chen , Tomasselli Alfredo G. , Heinrikson Robert L.
Published: July 2002

Apoptosis, or programmed cell death, plays a central role in the development and homeostasis of an organism. The breakdown of cellular proteins in apoptosis is mediated by caspases, which comprise a highly conserved family of cysteine proteases with specificity for aspartic acid residues at the P1 positions of their substrates. Multiple lines of evidence show that caspase‐9 is critical for an apoptosis pathway mediated via the mitochondria. In this study, the three‐dimensional structure of the catalytic domain of caspase‐9 and its interaction with the inhibitor acetyl‐Asp‐Val‐Ala‐Asp fluoromethyl ketone (Ac‐DVAD‐fmk) have been predicted by a segment matching modeling procedure. As expected, the predicted caspase‐9 structure shows both a high similarity in the overall folding topology and remarkable differences in the surface loop regions as compared to other caspase family members such as caspase‐1, ‐3 and ‐8, for which crystal structures have been determined. This kind of comparative analysis reflects the convergence–divergence duality among the caspases. Moreover, some subtle differences have been observed between caspase‐9 and caspase‐3 in the subsite contacts with the covalently linked inhibitor Ac‐DVAD‐fmk. Based on the X‐ray structural analysis of caspase‐8, a main chain carbonyl oxygen appears to be involved in a catalytic triad with the active site Cys and His residues. The corresponding carbonyl oxygen in caspase‐9, together with other expected features of the catalytic apparatus, appears in our model. The predicted structure of caspase‐9 can serve as a reference for subsite analysis relative to rational design of highly selective caspase inhibitors for therapeutic application.
In synergy with various cis‐acting elements, plant insterstitial telomere motifs regulate gene expression in Arabidopsis root meristems
Authors: Manevski Alexandra , Bertoni Georges , Bardet Claude , Tremousaygue Dominique , Lescure Bernard
Published: July 2002

The telo‐box, an interstitial telomere motif, was shown to regulate gene expression in root meristems, in synergy with a cis‐acting element involved in the activation of expression of plant eEF1A genes, encoding the translation elongation factor EF1A, and of several ribosomal protein genes. We demonstrate here that the telo‐box is also required for transcription activation by two other cis elements present within the promoter of genes encoding the acidic ribosomal protein rp40 and the proliferating cell nuclear antigen respectively. The control of gene expression by telo‐boxes during cell cycle progression in Arabidopsis root meristems is discussed. A parallel is drawn with the function of telomeric sequences in Saccharomyces cerevisiae.
H+/Ca2+ exchange driven by the plasma membrane Ca2+‐ATPase of Arabidopsis thaliana reconstituted in proteoliposomes after calmodulin‐affinity purification
Authors: Luoni Laura , Bonza Maria Cristina , De Michelis Maria Ida
Published: July 2002

The plasma membrane Ca2+‐ATPase was purified from Arabidopsis thaliana cultured cells by calmodulin (CaM)‐affinity chromatography and reconstituted in proteoliposomes by the freeze‐thaw sonication procedure. The reconstituted enzyme catalyzed CaM‐stimulated 45Ca2+ accumulation and H+ ejection, monitored by the increase of fluorescence of the pH probe pyranine entrapped in the liposomal lumen during reconstitution. Proton ejection was immediately reversed by the protonophore FCCP, indicating that it is not electrically coupled to Ca2+ uptake, but it is a primary event linked to Ca2+ uptake in the form of countertransport.
In vitro glucuronidation of xanthohumol, a flavonoid in hop and beer, by rat and human liver microsomes
Authors: Yilmazer M. , Stevens J.F. , Buhler D.R.
Published: July 2002

Xanthohumol (XN) is the major prenylated flavonoid of hop plants and has been detected in beer. Previous studies suggest a variety of potential cancer chemopreventive effects for XN, but there is no information on its metabolism. The aim of this study was to investigate in vitro glucuronidation of XN by rat and human liver microsomes. Using high‐performance liquid chromatography, two major glucuronides of XN were found with either rat or human liver microsomes. Release of the aglycone by enzymatic hydrolysis with β‐glucuronidase followed by liquid chromatography/mass spectrometry and nuclear magnetic resonance analysis revealed that these were C‐4′ and C‐4 monoglucuronides of XN.
APAP, a sequence‐pattern recognition approach identifies substance P as a potential apoptotic peptide
Authors: del Rio Gabriel , Castro-Obregon Susana , Rao Rammohan , Ellerby H.Michael , Bredesen Dale E.
Published: July 2002

We have previously described a novel cancer chemotherapeutic approach based on the induction of apoptosis in targeted cells by homing pro‐apoptotic peptides. In order to improve this approach we developed a computational method (approach for detecting potential apoptotic peptides, APAP) to detect short PAPs, based on the prediction of the helical content of peptides, the hydrophobic moment, and the isoelectric point. PAPs are toxic against bacteria and mitochondria, but not against mammalian cells when applied extracellularly. Among other peptides, substance P was identified as a PAP and subsequently demonstrated to be a pro‐apoptotic peptide experimentally. APAP thus provides a method to detect and ultimately improve pro‐apoptotic peptides for chemotherapy.
Intracellular molecular interactions of antitumor drug amsacrine (m‐AMSA) as revealed by surface‐enhanced Raman spectroscopy
Authors: Chourpa Igor , Morjani Hamid , Riou Jean-François , Manfait Michel
Published: July 2002

Cytotoxicity of several classes of antitumor DNA intercalators is thought to result from disturbance of DNA metabolism following trapping of the nuclear enzyme DNA topoisomerase II as a covalent complex on DNA. Here, molecular interactions of the potent antitumor drug amsacrine (m‐AMSA), an inhibitor of topoisomerase II, within living K562 cancer cells have been studied using surface‐enhanced Raman (SER) spectroscopy. The work is based on data of the previously performed model SER experiments dealing with amsacrine/ DNA, drug/topoisomerase II and drug/DNA/topoisomerase II complexes in aqueous buffer solutions. The SER data indicated two kinds of amsacrine interactions in the model complexes with topoisomerase II alone or within ternary complex: non‐specific (via the acridine moiety) and specific to the enzyme conformation (via the side chain of the drug). These two types of interactions have been both revealed by the micro‐SER spectra of amsacrine within living K562 cancer cells. Our data suppose the specific interactions of amsacrine with topoisomerase II via the side chain of the drug (particular feature of the drug/topoisomerase II and ternary complexes) to be crucial for its inhibitory activity.
Assembly of a cytoplasmic membrane protein in Escherichia coli is dependent on the signal recognition particle
Authors: de Gier Jan-Willem L. , Mansournia Parvaneh , Valent Quido A. , Phillips Gregory J. , Luirink Joen , von Heijne Gunnar
Published: July 2002

Targeting of the cytoplasmic membrane protein leader peptidase (Lep) and a Lep mutant (Lep‐inv) that inserts with an inverted topology compared to the wild‐type protein was studied in Escherichia coli strains that are conditional for the expression of either Ffh or 4.5S RNA, the two components of the E. coli SRP. Depletion of either component strongly affected the insertion of both Lep and Lep‐inv into the cytoplasmic membrane. This indicates that SRP is required for the assembly of cytoplasmic membrane proteins in E. coli.
Tissue‐specific subunit of the mouse cytosolic chaperonin‐containing TCP‐11
Authors: Kubota Hiroshi , Hynes Gillian M , Kerr Shona M , Willison Keith R
Published: July 2002

We have cloned a novel Tcp‐1‐related mouse testis cDNA encoding a polypeptide of 531 amino acids which shares 81.2% identity with the ζ subunit of the mouse cytosolic chaperonin‐containing TCP‐1 (CCT). Immunoblot analysis of mouse testis CCT subunits separated by 2‐dimensional gel electrophoresis indicates that this novel gene, Cctz‐2, encodes a CCT subunit of M
r 57 000 and pI 7.1. Cctz‐2 mRNA is detected only in testis whereas the other Cctz gene, Cctz‐1, is expressed in all tissues investigated. The CCTζ‐2 subunit may have specific functions in the folding of testicular proteins and for interactions with testicular molecular chaperones.
A single point mutation (E166Q) prevents dicyclohexylcarbodiimide binding to the photosystem II subunit CP29
Authors: Pesaresi Paolo , Sandonà Dorianna , Giuffra Elisabetta , Bassi Roberto
Published: July 2002

Energy‐dependent quenching of chlorophyll fluorescence (qE) reflects the action of a powerful mechanism of protection from photoinhibition in which the low pH in the chloroplast lumen induces dissipation of excess excitation energy. Dicyclohexylcarbodiimide (DCCD), a protein‐modifying agent, is a powerful inhibitor of qE and has been shown to react with acidic residues, in a hydrophobic environment, involved in proton translocation. The CP29 subunit of photosystem II has been proposed to be the site of qE quenching and shown to bind DCCD. We have hypothesised, on the basis of the CP29 protein sequence and of the structure of light‐harvesting complex II protein, that glutamic acid 166 is the DCCD binding site. In this study, we have produced recombinant proteins either with wild‐type sequence or carrying a mutation on the 166 position. We show that the mutant protein does not bind DCCD. This identifies E166 as the site whose protonation may lead to a conformational change triggering qE.
Role of dopamine at the onset of pupal diapause in the cabbage armyworm Mamestra brassicae
Authors: Noguchi Hirofumi , Hayakawa Yoichi
Published: July 2002

Experiments were conducted to examine the relationship between an onset of diapause and dopamine (DA) content in the cabbage armyworm Mamestra brassicae during pupation. The DA levels were significantly higher in haemolymph, integument and brain‐central nervous system of diapause‐destined pupae than in non‐diapause‐destined pupae. The elevated level of the integumental DA content was demonstrated to be due to an increase in dopa decarboxylase activity in diapausing pupal integuments through an enhancement of transcript levels of this enzyme. Elevation of the DA level accomplished by feeding l‐DOPA to last instar larvae induced a diapause‐like state in more than 50% of the pupae under long daylengths.
Antioxidant role of melatonin in lipid peroxidation of human LDL
Authors: Abuja Peter M. , Liebmann Peter , Hayn Marianne , Schauenstein Konrad , Esterbauer Hermann
Published: July 2002

It is concluded that melatonin is not incorporated into LDL in sufficient concentrations to prevent lipid peroxidation effectively. When melatonin is present in the incubation medium during oxidation, a partitioning equilibrium between aqueous and lipid phase is established. Only under these conditions can melatonin act as a chain breaking antioxidant. The concentrations required, however, are far beyond those found in human plasma. Therefore, the data in this study do not support a direct physiological relevance of melatonin as an antioxidant in lipid peroxidation processes.
Association of DNA with nuclear matrix in in vitro assembled nuclei induced by rDNA from Tetrahymena shanghaiensis in Xenopus egg extracts
Authors: Chen Ying , Zhang Bo , Li Xiufen , Zhai Zhonghe
Published: July 2002

Nuclei assembled in vitro from Xenopus egg extracts and DNA display many properties known from in vivo nuclei. However, the distribution pattern of DNA in such nuclei remains unknown. We introduced rDNA from Tetrahymena macronuclei into Xenopus egg extracts and examined the association of specific DNA sequences with the nuclear matrix(NM) of the nuclei formed. We found that the 5′ non‐transcribed spacers (5′‐NTS) specifically bound to the NM as previously shown in normal Tetrahymena macronuclei.
Different expression of adenylyl cyclase isoforms after retinoic acid induction of P19 teratocarcinoma cells
Authors: Lipskaia Larissa , Djiane Alexandre , Defer Nicole , Hanoune Jacques
Published: July 2002

We have investigated the adenylyl cyclase (AC) activity and gene expression in retinoic acid (RA)‐primed murine P19 teratocarcinoma cells, which recapitulate in vitro the first stages of neuroectodermal formation. Here we show that the P19 stem cells possess a basal Ca2+/CaM‐stimulated AC activity, which increases about 10‐fold after RA induction. The rise of AC activity is associated with a stage‐specific up‐regulation of AC2, AC5 and AC8 mRNAs and a down‐regulation of AC3 mRNA. P19 cells provide a powerful model to investigate the role and specific regulation of AC isoforms during neuronal differentiation.
c‐di‐GMP‐binding protein, a new factor regulating cellulose synthesis in Acetobacter xylinum
Authors: Weinhouse Haim , Sapir Shai , Amikam Dorit , Shilo Yehudit , Volman Gail , Ohana Patricia , Benziman Moshe
Published: July 2002

A protein which specifically binds cyclic diguanylic acid (c‐di‐GMP), the reversible allosteric activator of the membrane‐bound cellulose synthase system of Acetobacter xylinum, has been identified in membrane preparations of this organism. c‐di‐GMP binding is of high affinity (K
D 20 nM), saturable and reversible. The equilibrium of the reaction is markedly and specifically shifted towards the binding direction by K+. The c‐di‐GMP binding protein, structurally associated with the cellulose synthase, appears to play a major role in modulating the intracellular concentration of free c‐di‐GMP and thus may constitute an essential factor in regulating cellulose synthesis in vivo.
In vitro evidence that hsp90 contains two independent chaperone sites
Authors: Young Jason C , Schneider Christine , Hartl F.Ulrich
Published: July 2002

Hsp90 is an abundant and constitutively expressed stress protein and molecular chaperone. Here we dissected human hsp90 into three major domains to identify the putative chaperone site at which hsp90 binds unfolded polypeptide. Surprisingly, both the N‐terminal and the C‐terminal domain of hsp90 prevent the aggregation of denatured polypeptides. The chaperone activity of the N‐domain is inhibited by geldanamycin, a specific inhibitor of hsp90‐mediated protein refolding. While both domains suppress protein aggregation, only the C‐domain binds an antigenic peptide derived from VSV G. Based on these results, hsp90 may be the first chaperone to contain two independent chaperone sites with differential specificity.
Non‐balanced mix of mitochondrial DNA in cloned cattle produced by cytoplast‐blastomere fusion
Authors: Steinborn Ralf , Zakhartchenko Valeri , Wolf Eckhard , Müller Mathias , Brem Gottfried
Published: July 2002

We have investigated the transmission of parental mitochondrial DNA (mtDNA) in three clones of born cattle obtained by intraspecific cytoplast‐blastomere fusion. Using allele‐specific TaqMan PCR a low level transmission of blastomere mtDNA (DB mtDNA) into the cloned offspring was detected, thereby generating a heteroplasmic population of mtDNA. The amount of DB mtDNA was 13% and 18% in two animals of a clone which derived from a 24‐cell morula and 0.6% and 0.4% in two calves of clonal origin derived from a 92‐cell morula. These values are in accordance with the tendency expected for neutral mtDNA segregation that the fewer cell divisions that have occurred in the donor embryo, the higher the amount of DB mtDNA. We also found a strong decrease of DB mtDNA which was about three orders of magnitude in the third clone derived from a 52‐cell morula stage.
Engineering of chicken avidin: a progressive series of reduced charge mutants
Authors: Marttila Ari T , Airenne Kari J , Laitinen Olli H , Kulik Tikva , Bayer Edward A , Wilchek Meir , Kulomaa Markku S
Published: July 2002

Avidin, a positively charged egg‐white glycoprotein, is a widely used tool in biotechnological applications because of its ability to bind biotin strongly. The high pI of avidin (∼10.5), however, is a hindrance in certain applications due to non‐specific (charge‐related) binding. Here we report a construction of a series of avidin charge mutants with pIs ranging from 9.4 to 4.7. Rational design of the avidin mutants was based on known crystallographic data together with comparative sequence alignment of avidin, streptavidin and a set of avidin‐related genes which occur in the chicken genome. All charge mutants retained the ability to bind biotin tightly according to optical biosensor interaction analysis. In most cases, their thermal stability characteristics were indistinguishable from those of the wild‐type avidin. Our results demonstrate that the charge properties of avidin can be modified without disturbing the crucial biotin‐binding activity.