bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of a deletion mutant of the prosegment of human prorenin in Chinese hamster ovary cells
Authors: Nagahama Masami , Nakayama Kazuhisa , Hori Hitoshi , Murakami Kazuo
Published: July 2002

Expression plasmids encoding native human preporenin and a mutant deleted in its entire prosegment were transfected into Chinese hamster ovary cells. The cells transfected with the expression plasmid of native preporenin secreted exclusively inactive prorenin, while the cells transfected with the mutant secreted the active enzyme. The secreted amount of renin from the latter cells was much lower than that of prorenin from the former ones, although these two enzymes had little difference in specific activity after trypsin activation. These results suggest that the prosegment plays an important role in the secretory process of renin, although the fully active enzyme can be formed in its absence.
Rapid proton release during flash‐induced oxidation of cytochrome c oxidase
Authors: Nilsson Thomas , Hallén Stefan , Oliveberg Mikael
Published: July 2002

Protolytic reactions during the reaction between fully reduced cytochrome oxidase and oxygen have been investigated using the flow‐flash methodology. Enzyme reconstituted into phospholipid vesicles has been used and pH changes in the extravesicular medium have been detected with the pH indicator Phenol red. The results show the release of protons to the medium with a rate of 9.102 s−1 during reoxidation. The amplitude of the absorption change corresponds to at least one proton per functional unit of cytochrome oxidase.
[Hyp3]‐bradykinin and [Hyp3]‐Lys‐bradykinin interact with B2‐bradykinin receptors and stimulate inositol phosphate production in cultured human fibroblasts
Authors: Dengler Robert , Faußner Alexander , Müller-Esterl Werner , Roscher Adelbert A.
Published: July 2002

The recently isolated, naturally occurring peptide hormones [Hyp3]‐bradykinin and [Hyp3]‐Lys‐bradykinin were investigated for their agonist activity on solubilized binding sites from human fibroblasts. Both ligands competed with [3H]bradykinin binding in a dose‐dependent fashion with potencies similar to bradykinin (BK) and Lys‐BK. Biological activity was assessed by determination of inositol phosphate accumulation and cyclic 3',5'‐adenosine monophosphate synthesis in intact cultured cells. Stimulation by the hydroxylated peptides resulted in a pronounced accumulation of both parameters with similar effectiveness as BK and Lys‐BK. These results indicate that [Hyp3]‐BK and [Hyp3]‐Lys‐BK are agonists at the bradykinin receptor system with properties comparable to their non‐hydroxylated analogues. This suggests that hydroxylation of kinins does not alter receptor interaction or signal transduction in cultured human fibroblasts.
Domain structure of myosin subfragment‐1
Authors: Levitsky Dmitrii I. , Khvorov Nikolai V. , Shnyrov Valery L. , Vedenkina Natalia S. , Permyakov Eugene A. , Poglazov Boris F.
Published: July 2002

The structure of the myosin subfragment‐1 (SI) from rabbit skeletal muscle was studied using differential scanning microcalorimetry. Three independently melting regions (domains) were revealed in S1. Selective denaturation of the middle 50 kDa segment of the S1 heavy chain resulted in the disappearance of the heat sorption peak corresponding to the melting of the first, the most thermolabile domain without any effect on the thermally induced blue shift of the intrinsic tryptophan fluorescence spectrum which occurs within the temperature region of melting of the second domain. It is concluded that the most thermolabile domain seems to correspond to the N‐terminal part of the 50 kDa segment devoid of tryptophan residues.
One amino acid change in rat SMR1 polypeptide induces a 1 kDa difference in its apparent molecular mass determined by electrophoretic analysis
Authors: Rosinski-Chupin Isabelle , Rougeon François
Published: July 2002

SMR1 is a male‐specific, 19 kDa, in vitro translation product of Wistar rat submaxillary glands, which may be the precursor of a small hormone resembling the TRH. In Sprague‐Dawley and Fischer rats, instead of SMR1, a male‐specific 18 kDa polypeptide may be found. We have cloned the cDNA encoding the 18 kDa polypeptide. We show that the 19 and the 18 kDa polypeptides have the same sequence except for one amino acid change.
CTP‐dependent endogenous ADP‐ribosylation of a 38 kDa protein in HL‐60 cell membranes
Authors: Morinaga Naoko , Noda Masatoshi , Kato Iwao
Published: July 2002

Incubation of membranes of human promyelocytic leukemia HL‐60 cells with P2P]NAD led to ADP‐ribosylation of several proteins including a 38 kDa protein by endogenous ADP‐ribosyltransferases. The ADP‐ribosylation of the 38 kDa protein was distinctly different from others on the basis of pH dependency and heat stability at 50°C, suggesting that there are at least two endogenous ADP‐ribosyltransferases. It was enhanced by CTP, but not affected by ATP, GTP and UTP, whereas it was inhibited by GTPγS. [α‐32P]CTP bound to the 38 kDa protein immobilized on a nitrocellulose sheet, indicating that the 38 kDa protein which bound CTP is strongly ADP‐ribosylated by an endogenous ADP‐ribosyltransferase.
Effect of antipsychotic drugs on the molecular action of cholera toxin in rabbit intestinal epithelial cells
Authors: Longbottom David , van Heyningen Simon
Published: July 2002

Antipsychotic drugs of known antidiarrhoeal and anticalmodulin activity inhibited the cholera‐toxin‐catalysed ADP‐ribosylation of proteins of M
r, 37000,40000 and 45000 (thought to be regulatory components of the adenylate cyclase complex) that was previously shown to occur in plasma membranes from rabbit intestinal epithelial cells [(1989) Biochim. Biophys. Acta 1014, 289‐297]. There was no obvious correlation between the different activities of the drugs. The drugs also inhibited adenylate cyclase activity, but in this case the inhibition correlated well with the known IC50 values of the drugs for anticalmodulin activity and with their antidiarrhoeal activities.
Stereospecificity in enzymic and non‐enzymic oxidation of β‐O‐4 lignin model compounds
Authors: Jönsson Leif , Karlsson Olov , Lundquist Knut , Nyman Per Olof
Published: July 2002

The degradation of the erythro and threo isomers of the non‐phenolic lignin model compound 2‐(2,6‐dimethoxyphenoxy)‐1‐(3,4‐dimethoxyphenyl)‐1,3‐propanediol was examined. Enzymic and non‐enzymic oxidation of the diastereomers was performed with Trametes versicolor lignin peroxidase and cerium(IV) ammonium nitrate, respectively. Mixtures of approximately equal amounts of the diastereomers were partially degraded and subsequently analyzed with TLC and 1H‐NMR. Analysis of reaction mixtures from enzymic as well as non‐enzymic oxidation, revealed a preferential degradation of the threo form. Preliminary analyses of enzymic reaction mixtures of either the erythro or threo isomer suggest they yield in part different products. The observations made would have implications for the understanding of how enzymes attack lignins. They should also be taken into consideration in experiments where model compounds are being used to mimic native lignin.
Chloroplast ribosomal protein L32 is encoded in the chloroplast genome
Authors: Yokoi Fumiaki , Vassileva Anelia , Hayashida Nobuaki , Torazawa Keita , Wakasugi Tatsuya , Sugiura Masahiro
Published: July 2002

The 50 S subunit of chloroplast ribosomes was prepared from tobacco leaves. The proteins were fractionated and the N‐terminal amino acid sequence of a 14 kDa protein was determined. This sequence matches the N‐terminal sequence deduced from ORF55 located between ndhF and trnL on the small single‐copy region of tobacco chloroplast DNA. The deduced protein shows homology to E. coli and B. stearothermophilus L32 proteins, and it has been named as CL32 and ORF55 as rpl32. The tobacco chloroplast genome therefore contains 21 different ribosomal protein genes.
Galanin evokes a cytosolic calcium bursting mode and hormone release in GH3/B6 pituitary cells
Authors: Guérineau Nathalie , Drouhault Robert , Corcuff Jean-Benoît , Vacher Anne-Marie , Vilayleck Nidaravone , Mollard Patrice
Published: July 2002

The effects of galanin on secretion and cytosolic free Ca2+ concentration ([Ca2+
i) have been studied in GH3/B6 pituitary cells. Prolactin (PRL) and growth hormone (GH) release was measured in column Perifusion experiments; [Ca2+]i was monitored in single cells by dual emission microspectrofluorimetry using indo‐1 as intracellular Ca2+ probe. Galanin (0.1–1 nM) caused PRL and GH release coincident with a modest rise in [Ca2+]i The increase in [Ca2+]i comprises the establishment of characteristic long‐lasting bursts of [Ca2+]i transients. Galanin acts on Ca2+ entry through voltage‐gated Ca2+ channels since there was no response to the peptide when Cd2+ ‐ a Ca2+ channel blocker ‐ was added to the bath solution. The stimulation of bursting activity by galanin may provide a fine Ca2+ ‐signalling mechanism which maximally stimulates hormone release while avoiding refractory periods.
Calmodulin antagonists inhibit the phytochrome‐induced appearance of two nuclear encoded transcripts in radish cotyledons
Authors: Fourcroy Pierre , Pronost Stephane , Klein-Eude Dominique
Published: July 2002

The effects of two calmodulin antagonists on the phytochrome‐mediated appearance of two nuclear encoded transcripts in radish cotyledons have been investigated. The extent of inhibition of transcript accumulation was dependent of the time ellapsed between the administration of trifluoperazine and the light stimulus. When 1 mM trifluoperazine was administered to the seedlings 8 h before red light irradiation, the inhibition of transcript accumulation was up to 62% for the chlorophyll binding protein mRNA and 56% for the ribulose 1,5‐bisphosphate carboxylase small subunit mRNA. Similar results were obtained with W‐7 (0.1 mM).
A pseudosubstrate peptide inhibits protein kinase C‐mediated phosphorylation in permeabilized Rat‐1 cells
Authors: Eichholtz Thomas , Alblas Jacqueline , van Overveld Merian , Moolenaar Wouter , Ploegh Hidde
Published: July 2002

Activation of protein kinase C (PKC) in Rat‐1 fibroblasts leads to rapid phosphorylation of an 80‐kDa protein, a major substrate of PKC. Digiton‐in‐permeabilized cells perfectly supported this early response. Introduction of a PKC pseudosubstrate peptide inhibited 80 kDa phosphorylation with an IC50 of 1 μM, while a control peptide had no effect. The results indicate that this semi‐intact cell system can be used in combination with the inhibitory pseudosubstrate peptide to study the involvement of PKC in cellular processes.
Peptidyl‐prolyl cis‐trans isomerase does not affect the Pro‐43 cis‐trans isomerization rate in folded calbindin D9k
Authors: Kördel Johan , Drakenberg Torbjörn , Forsén Sture , Thulin Eva
Published: July 2002

The calcium‐binding protein calbindin D9k has previously been shown to exist in two folded forms only differing in the proline cis‐trans isomerism of the Gly‐42‐Pro‐43 amide bond. This bond is located in a flexible loop connecting the two EF‐hand Ca2+ sites. Calbindin D9k therefore constitutes a unique test case for investigating if the recently discovered enzyme peptidyl‐prolyl cis‐trans isomerase (PPIase) can affect the cis‐trans exchange rate in a folded protein. The 1H NMR saturation transfer technique has been used to measure the rate of interconversion between the cis and trans forms of calbindin in the presence of PPIase (PPIase:calbindin concentration ratio 1:10) at 35°C. No rate enhancement could be detected.
Organization of rat uricase chromosomal gene differs greatly from that of the corresponding plant gene
Authors: Motojima Kiyoto , Goto Sataro
Published: July 2002

The rat chromosomal gene for uricase was cloned. The gene spans more than 20 kilobases and the coding region is divided into 8 exons by 7 introns. The organization of the rat uricase gene is so greatly different from that of the soybean gene that the difference may not have been caused only by the removal of some ancestral introns during the period of widely separated evolution.
NaF and mononucleotides as inhibitors of 3'‐5'‐exonuclease activity and stimulators of polymerase activity of E. coli DNA polymerase I Klenow fragment
Authors: Potapova I.A. , Nevinsky G.A. , Khomov V.V. , Lavrik O.I.
Published: July 2002

It has been shown that, in the absence of dATP in the poly(dT) · oligo(dA) template‐primer complex, the rate of primer cleavage by the E. coli DNA polymerase I Klenow fragment equals 4% of polymerization rate, while in the presence of dATP it equals as much as 50–60%. NaF and NMP taken separately inhibit exonuclease cleavage of oligo(dA) both with and without dATP. The addition of NaF (5–10 mM) or NMP (5–20 mM) increases the absolute increment of polymerization rate 5–9‐fold relative to the absolute decrement of the rate of nuclease hydrolysis of primer. This proves the assumption that not more than 10–20% of primer molecules, interacting with the exonuclease center of polymerase, are cleaved by the enzyme. Presumably, NaF and nucleotides disturb the coupling of the 3'‐end of oligonucleotide primer to the exonuclease center of the enzyme. As the primers mostly form complexes with the polymerizing center, the reaction of polymerization is activated.
Two‐dimensional crystallization of reaction centers from Chloroflexus aurantiacus
Authors: Barnakov A.N. , Demin V.V. , Kuzin A.P. , Zargarov A.A. , Zolotarev A.S. , Abdulaev N.G.
Published: July 2002

Two‐dimensional crystals of photosynthetic reaction centers from Chloroflexus aurantiacus were obtained from protein‐lipid‐detergent micelles by detergent dialysis. The size of crystals was up to 2 μm. Some of them were multilayered crystals. However, other crystal forms were also observed. Preliminary image processing analysis showed that crystals of one crystal form referred to two‐sided plane group p2 and had the following unit cell parameters: a = 17,6 nm, b = 18,0 nm, γ = 84°. The contour map of the crystal stain‐excluding region was calculated by the Fourier‐filtering procedure at about 2 nm resolution.
Nucleotide sequence of four genes encoding ribosomal proteins from the ‘S10 and spectinomycin’ operon equivalent region in the archaebacterium Halobacterium marismortui
Authors: Arndt Evelyn
Published: July 2002

Four genes encoding ribosomal proteins HmaS17, HmaL14, HmaL24 and HS3, have been identified in the λEMBL3 clone PP∗7 from a genomic library of the archaebacterium Halobacterium marismortui. The clone contains genes from the ‘S10 and spectinomycin’ operon equivalent region. Three of the deduced proteins are homologous to the corresponding Escherichia coli and Methancoccus vannielii S17, L14 and L24 proteins, as well as to eukaryotic proteins from rat or yeast. HS3 was identified as an extra protein corresponding to the gene product for orfc in M. vannielii and the eukaryotic ribosomal protein RS4 from rat. The equivalence of HmaL24 (HL16) and E. coli L24, which share only 28% identical amino acid residues, could now be shown by localizing the HmaL24 gene at the same position in the cluster.
TPA and resiniferatoxin‐mediated activation of NADPH‐oxidase
Authors: Evans A.T. , Sharma P. , Ryves W.J. , Evans F.J.
Published: July 2002

The non‐tumour promoting irritant, resiniferatoxin, was capable of activating the NADPH‐oxidase respiratory burst of starch‐elicited, but not resident mouse peritoneal macrophages in vitro. Unlike TPA, the response was synergised by incubation with zymosan. The Rx‐stimulated NADPH‐oxidase activity in a cell‐free assay was selectively enhanced in the presence of exogenous Rx‐kinase rather than PKC and in the absence of Ca2+. Since resiniferatoxin is a poor activator of PKC, it is probable that the Ca2+‐independent Rx‐kinase plays a role in activation of the macrophage respiratory burst following stimulation by zymosan.
Modelling heme d1
Authors: Andersson Laura A. , Loehr Thomas M. , Wu Weishih , Chang Chi K. , Timkovich Russell
Published: July 2002

The heme d
1 macrocycle of Ps. aeruginosa dissimilatory nitrite reductase is an iron porphyrin‐3,8‐dione with a 17‐acrylate substituent. We have compared the RR properties of Cu‐d
1, the copper(II) TME of extracted heme d
1, with those of models that differ only with respect to the acrylate: Cu‐17‐acrylate‐mesoporphyrin‐3,8‐dione (2) and Cu‐mesoporphyrin‐3,8‐dione (3). The RR spectrum of Cu‐d
1 is very similar to that of 2, including v(C=O) at ~ 1720 cm−1. Replacement of the acrylate with propionate changes the spectrum markedly. For example, the v(C=O) mode of 3 shifts to 1712 cm−1, and peaks of Cu‐d
1 and 2 at ~ 1400 and ~ 1535 cm−1 are shifted or absent from the spectrum of 3. FTIR spectra of 2 and 3 also differ in their νoxo(C=O) frequencies. The acrylate thus has a surprisingly strong influence on the electronic structural and spectral properties of heme d
1. These data provide a foundation for studies of the novel biological porphyrindione macrocycles.
Epidermal growth factor induces tyrosine phosphorylation of epidermal growth factor receptors not occupied with ligand in intact A431 cells
Authors: Sorokin A.B. , Reshetnikova G.F. , Kudryavtseva N.V. , Nikolsky N.N.
Published: July 2002

The mechanism of epidermal growth factor receptor (EGF‐R) autophosphorylation in intact A431 cells was studied. We detected epidermal growth factor (EGF) induced tyrosine phosphorylation of EGF‐R not occupied with ligand. Cell monolayers were subjected to irradiation after incubation with photoreactive derivative of EGF and uncoupled EGF was extracted by acidic treatment. Subsequent immunoprecipitation with antiphosphotyrosine antibodies resulted in precipitation of both EGF‐R complexes with EGF and EGF‐R with unoccupied ligand‐binding site. The fact of precipitation of EGF‐R with unoccupied ligand‐binding site in conjunction with our finding of rapid dephosphorylation of EGF‐R after EGF extraction by acidic treatment, strongly supports the interpretation that cross‐phosphorylation of EGF‐R may take place in intact cells.
Potassium channels in basolateral membrane vesicles from pars convoluta of rabbit proximal tubule
Authors: Jacobsen Christian , Blokkebak-Poulsen Jens , Sheikh M.I.
Published: July 2002

The characteristics of 86Rb+ fluxes through conductive channels in basolateral‐membrane vesicles isolated from pars convoluta of rabbit proximal tubule were investigated. In KCl loaded vesicles a transient accumulation of 86Rb+ was observed which was inhibited by BaCl2. The accumulation was driven by an electrical diffusion potential, as shown in experiments using membrane vesicles loaded with Li2SO4 and an outwardly directed Li+ gradient established with a Li+‐ionophore. The vesicles containing the channel showed a cation selectivity with the order K+ = Rb+ < < Li+ ⩾ Na+ < choline+. The 86Rb+ flux was dependent on intravesicular Ca2+. Increasing concentrations of Ca2+ gradually decreased the 86Rb+ uptake.
Solubilization of sarcoplasmic reticulum membranes by sodium dodecylsulphate
Authors: Prado A. , Muga A. , Castresana J. , Goñi F.M. , Arrondo J.L.R.
Published: July 2002

In order to improve our understanding of membrane protein solubilization by sodium dodecylsulphate, sarcoplasmic reticulum vesicles have been treated with this surfactant at different detergent: protein mole ratios. Effects on Ca2+‐ATPase activity, membrane protein solubilization, and protein conformation have been independently monitored, and correlations among the various parameters have been observed. The thermal denaturation of sarcoplasmic reticulum proteins in the presence of sodium dodecylsulphate has also been characterized spectroscopically.
Is cytochrome P‐450scc a transmembrane protein?
Authors: Usanov Sergey A. , Chernogolov Alexey A. , Chashchin Vadim L.
Published: July 2002

The topology of cytochrome P‐450scc in the inner mitochondrial membrane of adrenal cortex has been investigated using monospecific antibodies to cytochrome P‐450scc and its fragments F1 (Ile1‐Arg250), F2 (Asn257‐Ala481) and F3 (Asn257‐Arg399). Antibodies to F1 and F2 were shown to effectively bind to the matrix and cytosolic sides of the inner membrane. Antibodies to F3 specifically interacted only with the matrix side of the membrane. These data are consistent with a model of molecular organization which shows that cytochrome P‐450scc is a transmembrane protein, both N‐ and C‐terminal sequences of the cytochrome being able to span the membrane.
Catecholamines inhibit steroidogenesis by cultured porcine thecal cells
Authors: Morley Paul , Calaresu Franco R. , Armstrong David T.
Published: July 2002

The ovaries of many species contain catecholamines and β‐adrenergic receptors. The present studies were done to determine if catecholamines play a role in the regulation of androgen production by porcine theca cells. Basal and luteinizing hormone (LH)‐stimulated androstenedione production was significantly inhibited by noradrenaline and isoproterenol. The inhibitory effects were dose‐dependent and were enhanced when the cultures contained the carboxy‐O‐methyl transferase inhibitor, U‐0521. The inhibitory effect of isoproterenol was reversed by the β‐adrenergic antagonist, metoprolol. Isoproterenol caused a generalized inhibition of LH‐stimulated steroidogenesis, decreasing the accumulation of pregnenolone, progesterone, androstenedione and estradiol in the culture medium. These studies suggest that catecholamines may be important regulators of thecal androgen production.
Detoxification of the macrolide toxin brefeldin A by Bacillus subtilis
Authors: Kneusel Richard E. , Matern Ulrich , Wray Victor , Klöppel Klauss-Dieter
Published: July 2002

The macrolide brefeldin toxin A is a determinant of Alternaria leaf blight disease in safflower, which causes severe economic losses worldwide. Soilborne bacteria, classified as Bacillus subtilis spp., were isolated and shown to readily metabolize brefeldin A in laboratory culture to one major product. This product was identified by high resolution 2D 1H NMR and FAB mass spectroscopies as the acid resulting from hydrolysis of the macrolide ring in brefeldin A. In contrast to brefeldin A, the acid completely lacked phytotoxic activity in the standard leaf bioassay. Detoxification of brefeldin A by the lactonase activity from Bacillus subtilis may be exploited in the future to introduce resistance to Alternaria leaf blight in sufflower.
Effect of NAD coenzyme on the inactivation of glyceraldehyde‐3‐phosphate dehydrogenase by anionic phospholipids
Authors: Sidorowicz A. , Modrzycka T. , Gołebiowska J. , Siemieniewski H.
Published: July 2002

The inactivation of bovine heart glyceraldehyde‐3‐phosphate dehydrogenase by phosphatidylinositol (PI) and phosphatidylserine (PS) in the form of liposomes was investigated in the presence and absence of NAD excess. In the absence of NAD, the enzyme activity decreased to about 50% of its initial value at 0.6 mM PI and 0.8 mM PS (lipid‐to‐protein molar ratio 600 and 800, respectively). In the same lipid concentration range almost full regainment of the activity was observed in the presence of 80 μM NAD. It was shown that the excess of NAD protects the enzyme against conformational change induced by the phospholipids. Centrifugation experiments showed that both PI and PS bind significant amounts of NAD.
A Drosophila nuclear localisation signal included in an 18 amino acid fragment from the serendipity δ zinc finger protein
Authors: Noselli Stéphane , Vincent Alain
Published: July 2002

Sequence analysis of the nuclear Drosophila serendipity δ Cys‐2/His‐2 finger protein indicated the presence of a short motif of positively charged amino acids, with homology of the SV40 large T and c‐myc nuclear localisation signals. Using P‐element mediated transformation we constructed transgenic Drosophila lines expressing β‐galactosidase fusion proteins, containing (or not) an 18 residue segment of sry δ including this basic, PTKKRVK, motif. Histochemical detection of fusion proteins on dissected tissues showed that this segment of sry δ can act autonomously to drive the β‐galactosidase in nuclei.
Triiodide reduction by cellobiose: quinone oxidoreductase of Phanerochaete chrysosporium
Authors: Bao Wenjun , Renganathan V.
Published: July 2002

Cellobiose: quinone oxidoreductase (CBQase) in the presence of cellobiose inhibits peroxidase‐catalyzed oxidation of iodide to triiodide (I3,−). This inhibition is due to the two‐electron reduction of I−
3 by CBQase. The apparent Km
of I−
3 for this reaction is 120 μM and the specific activity is 57 μmol·min−1·mg−1. A proposed mechanism for I−
3 reduction by CBQase involves initial reduction of the flavin moiety by cellobiose to produce a dihydroflavin. This is followed by the substitution of one of the iodine atoms of I−
3 at the C(4a)‐position of dihydroflavin to generate C(4a)‐iododihydroflavin and two iodide ions. The C(4a)‐iododihydroflavin eliminates HI to regenerate the oxidized CBQase.
Geometrical correlations useful for design of sequence‐specific DNA narrow groove binding ligands
Authors: Zasedatelev A.S.
Published: July 2002

Isohelical geometry of sequence‐specific DNA narrow groove binding ligands was analyzed in terms of H‐bond donor/acceptor complementarily between the base pair atoms facing into the narrow groove and the corresponding H‐bond donating atoms regularly disposed along the ligand molecule. Spatial correlations found in analytical form were applied to analysis of naturally occurring and hypothetical drug molecule structures. For the case of B‐like isohelices the permitted value of the distance L
o between each two neighboring H‐bond donating atoms of the ligand as well as the bending angle τo of the line subsequently connecting these atoms were estimated as follows: L
o ⋍ (5.0±0.4) Å: τo ⋍ (26±2)*.
Purification of two thermostable components of messenger ribonucleoprotein particles (mRNPs) from Xenopus laevis oocytes, belonging to a novel class of RNA‐binding proteins
Authors: Deschamps Stéphane , Viel Alain , Denis Herman , le Maire Marc
Published: July 2002

We have purified and partially sequenced two proteins from Xenopus laevis previtellogenic oocytes, belonging to messenger ribonucleoprotein particles (mRNPs). The purification procedure rests on the thermostability of these proteins, which remain soluble after heating the cell extracts at 80°C. The thermostable proteins can be identified with two of the most abundant components (mRNP3 and mRNP4) of the mRNPs, described by Darnbrough and Ford (1981) [Eur. J. Biochem. 118, 415–424]. mRNP3 and mRNP4 are homologous to each other, but to no other protein of known sequence. The abundance and semi‐periodic distribution of proline residues in mRNF3 and mRNF4 sequences suggest that these RNA‐binding proteins adopt an unusual type of conformation
Production of a full length Tat protein in E. coli and its purification
Authors: Armengaud Jean , de Nuova Perez Lidia , Lemay Piere , Masson Jean-Michel
Published: July 2002

A full length tat gene was constructed by a combination of a polymerase chain reaction (PCR) for the first exon and chemical synthesis for the second exon. This gene was expressed in E. Coli under the control of the strongly regulated araB promoter, either directly or fused to a secretion signal encoding sequence. We the defined a rapid, three‐step procedure for the purification of the Tat protein
Assignment of the 15N NMR spectra of reduced and oxidized Escherichia coli thioredoxin
Authors: Chandrasekhar K. , Krause Günter , Holmgren Arne , Dyson H.Jane
Published: July 2002

As a necessary first step in the use of heteronuclear correlated spectra to obtain high resolution solution structures of the protein, assignment of the 15N NMR spectra of reduced and oxidized Escherichia coli thioredoxin (M
r 12 000) uniformly labeled with 15N has been performed. The 15N chemical shifts of backbone amide nitrogen atoms have been determined for both oxidation states of thioredoxin using 15N‐1H correlated and two‐dimensional heteronuclear single‐quantum coherence (HSQC) TOCSY and NOESY spectra. The backbone assignments are complete, except for the proline imide nitrogen resonances and include Gly33, whose amide proton resonance is difficult to observe in homonuclear 1H spectra. The differences in the 15N chemical shift between oxidized and reduced thioredoxin, which occur mainly in the vicinity of the two active site cysteines, including residues distant in the amino acid sequence which form a hydrophobic surface close to the active site, are consistent with the differences observed for proton chemical shifts in earlier work on thioredoxin.
Comparison of mammalian, chicken and Xenopus brain‐derived neurotrophic factor coding sequences
Authors: Isackson Paul J. , Towner Melvin D. , Huntsman Molly M.
Published: July 2002

We have used the polymerase chain reaction (PCR) to amplify and clone coding sequences of the mature region of brain‐derived neurotrophic factor (BDNF) from monkey, rat, chicken and Xenopus genomic DNA. Consistent with previous reports, the predicted amino acid sequences obtained in this manner from monkey and rat were identical to other mammalian BDNF sequences. The chicken and Xenopus BDNF sequences are also highly conserved, but contain 7 and 8 amino, acid substitutions, respectively, compared to mammalian BDNF. Comparison of these sequences with the homologous NGF and NT3 coding regions provides further insight into amino acid residues that may be responsible for the different receptor specificities of these factors.
Thyroid hormone increases glyceraldehyde 3‐phosphate dehydrogenase gene expression in rat liver
Authors: Ness Gene C. , Pendleton Laura C.
Published: July 2002

Livers from hypophysectomized rats had low levels of glyceraldehyde 3‐phosphate dehydrogenase mRNA. Administration of L‐triiodothyronine increased these levels over 20‐fold. The peak response was seen 72 h after hormone administration. A half‐maximal response was obtained with 5 μg of T3 per 100 g of body weight. Thus the expression of hepatic glyceraldehyde 3‐phosphate dehydrogenase appears to be regulated by thyroid hormone.
Interaction with the inositol 1,4,5‐trisphosphate receptor promotes Ca2+ sequestration in permeabilised insulin‐secreting cells
Authors: Islam Md.Shahidul , Nilsson Thomas , Rorsman Patrik , Berggren Per-Olof
Published: July 2002

Electropermeabilised insulin‐secreting RINm5F cells sequestered Ca2+, resulting in a steady‐state level of the ambient free Ca2+ concentration corresponding to 723 ± 127 nM (mean ± SEM, n = 10), as monitored by a Ca2+‐selective minielectrode. Inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) promoted a rapid and pronounced release of Ca2+. This Ca2+ was resequestered and a new steady‐state Ca2+ level was attained, which was always lower (460 ± 102 nM, n = 10, P < 0.001) than the steady‐state Ca2+ level maintained before the addition of Ins(1,4,5)P3. Whereas the initial reuptake of Ca2+ subsequent to Ins(1,4,5,)P3 stimulation was relatively slow, the later part of reuptake was fast as compared to the reuptake phases of a pulse addition of extraneous Ca2+. In the latter case the uptake of Ca2+ resulted in a steady‐state level similar to that found in the absence of Ins(1,4,5)P3. Addition of Ins(1,4,5)P3 under this condition resulted in a further Ca2+ uptake and thus a lower steady‐state Ca2+ level, Heparin, which binds to the Ins(1,4,5)P3 receptor, also lowered the steady‐state free Ca2+ concentration, In contrast to Ins(1,4,5)P3 inositol 1,3,4,5‐tetrakis‐phosphate was without effect on Ca2+ sequestration. These findings are consistent with the presence of a high‐affinity Ins(1,4,5)P3 receptor promoting continuous release of Ca2+ under basal conditions and/or the Ins(1,4,5)P3 receptor being actively involved in Ca2+ sequestration.
Factors contributing to the inhibition of HIV reverse transcriptase by chain‐terminating nucleotides in vitro and in vivo
Authors: Goody R.S. , Müller B. , Restle T.
Published: July 2002

Arguments are presented leading to the conclusion that two major factors contribute to the potency of inhibition of DNA‐polymerase activity by chain‐terminating nucleotides. The relative significance of these factors varies with the reaction conditions, particularly with the length of the template and the concentration ratio of enzyme (reverse transcriptase or other DNA polymerase) to primer. It is concluded that potent inhibition of HIV‐reverse transcriptase activity under typical in vitro and in vivo conditions arises from different features of the interaction of chain terminators with the enzyme. A new method of testing for the parameter important under in vivo conditions is suggested.
Biosynthesis of ferulic acid esters of plant cell wall polysaccharides in endomembranes from parsley cells
Authors: Meyer Knut , Kohler Annegret , Kauss Heinrich
Published: July 2002

A microsomal preparation from suspension‐cultured parsley cells is able to transfer ferulic acid from the respective CoA thioester to endogenous acceptors. The reaction is not enhanced by digitonin but stimulated by Mg2+, Ca2+ and Co2+. Spermine can partly replace divalent ions. Solubility properties and degradation by polysaccharide hydrolases suggest that the products are polymeric cell wall carbohydrates. Sucrose density gradient centrifugation revealed that the most active vesicle fraction is distinct from plasma membranes but does also not peak with IDPase. It is suggested that a subfraction of the Golgi‐apparatus is the source of enzyme and acceptors.
Non‐cognizable ribonucleotide, 2′AMP, binds to a mutant ribonuclease T1 (Y45W) at a new base‐binding site but not at the guanine‐recognition site
Authors: Hakoshima Toshio , Itoh Takeshi , Gohda Keigo , Tomita Ken-ichi , Uesugi Sei-ichi , Nishikawa Satoshi , Morioka Hiroshi , Ohtsuka Eiko , Ikehara Morio
Published: July 2002

Complex of a mutant ribonuclease T1 (Y4SW) with a non‐cognizable ribonucleotide, 2′AMP, has been determined and refined by X‐ray diffraction at 1.7 Å resolution. The 2′AMP molecule locates at a new base‐binding site which is remote from the guanine‐recognition site, where 2′GMP was found to be bound. The nucleotide adopts the anti conformation of the glycosidic bond and C3′‐exo sugar pucker. There exists a single hydrogen bond between the adenine base and the enzyme, and, therefore, the site found is apparently a non‐specific binding site. The results indicate that the binding of 2′AMP to the guanine‐recognition site is weaker than that to the new binding site.
Effects of lonidamine on the membrane electrical properties of Ehrlich ascites tumor cells
Authors: Santini Maria T. , Cametti Cesare , Indovina Pietro L. , Malorni Walter , Benassi Marcello , Gentile F.Paolo , Castiglione Susanna , Floridi Aristide
Published: July 2002

The effects of lonidamine on membrane electrical properties of Ehrlich ascites tumor cells are investigated. Using a dielectric relaxation technique based on the Maxwell‐Wagner effect and elaborated by a ‘single‐shell’ fitting procedure, the data indicate that both membrane conductivity and membrane permittivity increase after treatment of these cells with lonidamine while the conductivity of the cytosol remains unchanged. Changes in membrane proteins and/for lipids are suggested which lead to altered membrane structure and/or function.
Determination of the DNA binding site of the GAL4 protein A photo‐CIDNP study
Authors: Serikawa Yasuharu , Shirakawa Masahiro , Kyogoku Yoshimasa
Published: July 2002

After the assignment of 1H NMR signals of aromatic side chains by means of specific deuteration, we analyzed the DNA binding site of GAL4 by measuring photo‐CIDNP spectra. The results showed that Trp36 is involved in both the specific interaction with UASG and non‐specific DNA binding. This residue is located inside the Cys‐rich region, but outside the putative Zn‐finger. The photo‐CIDNP spectrum also showed that the side chains of Tyr40 and His53 are not exposed on the surface of the protein.
Biological activity of recombinant Ricinus communis agglutinin A chain produced in Escherichia coli
Authors: O'Hare Mary , Roberts Lynne M. , Lord J.Michael
Published: July 2002

DNA encoding Ricinus communis agglutinin A chain was listed into the E. coli expression vector pDS S/3. Induced E. coli 71.18 cells which had been transformed with this plasmid express Ricinus communis agglutinin A chain in a soluble and biologically active form. Recombinant Ricinus communis agglutinin A chain had ribosomal RNA N‐glycosidase activity and was approximately 10‐fold less active than ricin A chain in a cell‐free protein synthesis inhibition assay.
Isoproterenol‐stimulated labelling of particulate proteins by using [adenylate‐32P]NAD+ independent on a cAMP‐dependent protein kinase in parotid acinar ells
Authors: Sugiya Hiroshi , Hara-okoyama Miki , Furuyama Shunsuke
Published: July 2002

When saponin‐permeabilized rat parotid acinar cells were incubated with [adenylate‐32P]NAD+, labelling of proteins (33+ 27 and 23 kDa) in particulate fractions of the cells was stimulated by isoproterenol. The effect of isoproterenol was completely blocked by a β‐antagonist. Both forskolin or CAMP mimicked the effect of isoproterenol on the labelling. However, an inhibitor of cAMPdPK failed to induce complete inhibition of the effects of isoproterenol, forskolin and cAMP. When the labelled proteins were treated with snake venom phosphodiesterase, neither [32P]5′‐AMP nor [32P]phosphoribosyladenosine was released. These results suggest that covalent modification of proteins with NAD+, which is distinct from ADP‐ribosylation and cAMPdPK‐dependent phosphorylation, is coupled to β‐receptor‐cAMP signalling system in rat parotid acinar cells.
Effect of ploidy on transcription levels in cultured rat aortic smooth muscle cells
Authors: van Neck Johan W. , van Berkel Patrick H.C. , Telleman Pieter , Steijns Linda S.W. , Onnekink Carla , Bloemers Henri P.J.
Published: July 2002

Aging and hypertension increase the number of polyploid smooth muscle cells (SMC) in a blood vessel. We assessed the effect of ploidy on the transcription of several genes in SMC cultures derived from newborn and adult rats. In diploid and tetraploid subcultures of SMC from newborn rats, RNA expression of the genes assayed is linked with ploidy. However, when phenotypically different SMC cultures derived from newborn and adult rats were compared, transcription levels varied from gene to gene and not linked with the ploidy. Thus, differences in gene expression due to polyploidy are superimposed on those due to other phenotypical features.
Effects of 2(3)‐tert‐butyl‐4‐hydroxyanisole (BHA) on in situ mitochondria of Trypanosoma cruzi
Authors: Aldunate J. , Coloma-Torres L. , Spencer P. , Morello A. , Ojeda J.M. , Repetto Y.
Published: July 2002

Results obtained with in situ mitochondria or Trypanosoma cruzi showed that this protozoon had only two energy coupling sites, sites II and III that correspond to higher eukaryote mitochondria. Rotenone did not inhibit the oxygen uptake or the parasite. These results suggest that the NADH‐ubiquinone segment of the respiratory chain has no activity. Studies with in situ mitochondria confirmed that BHA, an antioxidant food additive, blocks the mitochondrial electron transport chain at the succinate‐cytochrome b segment being the molecular basis of this trypanocidal action.
Fluorescence study of the thyroxine‐dependent conformational changes in human serum transthyretin
Authors: González Gustavo , Tapia Guillermina
Published: July 2002

Fluorescence studies of transthyretin (TTR) were conducted to detect structural changes associated with the environment of its two tryptophans, induced by binding of thyroxine (T4). Non‐radiative tryptophans relaxation rate has an activation energy of 6.4 kcal/mol for TTR, which is decreased to 4.4 kcal/mol for TTR‐T4 complex. The maximum fluorescence wavelength was red‐shifted as the excitation wavelength was increased. T4 changed the magnitude of this shift. T4 binding per se changed the emission maximum reflecting different environments of the tryptophans. Double‐quenching experiments also showed that T4 produces changes in the tryptophans environments. These findings were interpreted as the result of structural alterations in the protein matrix induced by T4 which contribute in part to explain the negative cooperativity associated with the occupancy of the second binding site.
Conversion of 5,6‐dihydroxyeicosatetraenoic acids A novel pathway for lipoxin formation by human platelets
Authors: Tornhamre Susanne , Gigou Agnès , Edenius Charlotte , Lellouche Jean-Paul , Lindgren Jan Åke
Published: July 2002

Leukotriene A4 may be metabolized to 5(S),6(R)‐ and 5(S),6(S)‐dihydroxy‐7,9‐trans‐11,14‐cis‐eicosatetraenoic acids by enzymatic or non‐enzymatic hydrolysis. Incubation of human platelet suspensions with these dihydroxy acids led to the formation of lipoxin A4 and 6(S)‐lipoxin A4 via lipoxygenation at C‐15. Furthermore, human platelets converted the two 5(R),6(S)‐ and 5(R),6(R)‐dihydroxy‐7,9‐trans‐11,14‐cis‐eicosatetraenoic acids to tetraene‐containing trihydroxyeicosatetraenoic acids. In contrast, leukotrienes C4, D4 and E4 were not transformed to cysteinyl‐lipoxins, Time‐course studies of leukotriene A4 metabolism in human platelet suspensions indicated lipoxin formation via two pathways: (i) direct conversion of leukotriene A4, leading to formation of the lipoxin intermediate 15‐hydroxy‐leukotriene A4; and (ii) 15‐lipoxygenation of the 5(S),6(R)‐ and 5(S),6(S)‐dihydroxyeicosatetraenoic acids. The results demonstrate that lipoxygenation at C‐15 of 5,6‐dihydroxy‐7,9,11,14‐eicosatetraenoic acids may be an alternative novel pathway for platelet‐dependent lipoxin formation.
Purification of the MglC/E membrane proteins of the binding protein‐dependent galactose transport system of Salmonella typhimurium
Authors: Richarme Gilbert , Kohiyama Masamichi
Published: July 2002

The high affinity galactose transport system of consists of Salmonella typhimurium consists of four proteins, a periplasmic galactose binding protein (the MglB protein), and three inner membrane‐associated proteins, the MglA, MglC and MglE proteins. We purified the MglC/E proteins from an MglC/E hyperproducing strain after solubilisation of inclusion bodies in guanidine hydrochloride followed by renaturation in a detergent‐containing buffer and affinity chromatography on a MglB—Sepharose column. The MglC/E proteins are devoid of ATPase activity and they complement an extract from a strain carrying a plasmid with the mglA gene for reconstitution of the MglB‐dependent galactose transport in proteoliposomes.
Glycopeptide of P0 protein inhibits homophilic cell adhesion Competition assay with transformants and peptides
Authors: Yazaki Takahito , Miura Masayuki , Asou Hiroaki , Kitamura Kunio , Toya Shigeo , Uyemura Keiichi
Published: July 2002

Expression of major myelin glycoprotein P0 by PO cDNA transfection into C6 glioma cells promoted homophilic cell adhesion of the cells. After the dissociated cells were incubated for various times, the number of particles at each time point was measured. The total number of particles decreased to 24% in 60 min for transformant (C6P0) cells, in contrast to only 68% for control (C6P0′) cells. To confirm the homophilic mechanism of adhesion, mixed‐cell aggregation experiments were performed. Among the four synthetic peptides corresponding to a part of the P0 sequence used, only peptide 3 (residues 90–96), which contained a carbohydrate attaching site, caused considerable inhibition of cell aggregation (approximately 50%). In addition, the glycopeptide (residues 91–95) obtained from bovine P0 markedly inhibited cell aggregation (by approximately 85%).
Ca2+/calmodulin‐activated protein phosphatase
Authors: Nakamura Taro , Tsutsumi Hiroko , Mukai Hideyuki , Kuno Takayoshi , Miyakawa Tokichi
Published: July 2002

Protein phosphatase (PP2B) whose activity is stimulated 12‐20‐fold by Ca2+/calmodulin (CaM) was partially purified by CaM‐Sepharose and heparin‐agarose chromatographies from cell extract of the yeast Saccharomyces cerevisiae. PP2B activity was not detectable in a mutant in which two genes (CMP1 and CMP2) encoding homologs of mammalian PP2B catalytic subunit were disrupted. We have previously shown that the double gene disruption has no significant effect on the growth of yeast [1991, Mol. Gen. Genet. 227, 52–59]. The results indicated that CMP1 and CMP2 are the only genes that encode the PP2B catalytic polypeptide in S. cerevisiae,and PP2B activity is not essential for the growth of the yeast under normal conditions.
Peptidyl‐prolyl cis‐trans isomerase from Bacillus subtilis A prokaryotic enzyme that is highly sensitive to cyclosporin A
Authors: Herrler Michael , Bang Holger , Brune Kay , Fischer Gunter , Marahiel Mohamed A.
Published: July 2002

Cyclophylins are members of a class of proteins with peptidyl‐protyl cis‐trans isomerase activity. These enzymes bind the immunosuppressive agent, cyclosporin A (CsA), which acts as a competitive inhibitor. The peptidyl‐prolyl cis‐trans isomerase from Bacillus subtilis (PPlase) was purified to homogeneity in a 4‐step purification procedure, which resulted in a 100‐fold protein purification with a yield of 5%, Coomassie blue‐stained SDS‐PAGE revealed a single band of about 18 kDa. PPlase activity was determined using synthetic peptides as substrates in a 2‐step reaction coupled to chymotrypsin. Treatment of Bacillus subtilis PPlase by CsA revealed an inhibition constant of K
1=175 nM, which differs from cyclophilin of enterobacteria such as E. coli or Salmonella typhimurium and is in the range of human enzymes.
The genetic defect of the original Norwegian lecithin:cholesterol acyltransferase deficiency families
Authors: Skretting Grethe , Blomhoff Jan P. , Solheim Jorun , Prydz Hans
Published: July 2002

Three of the original Norwegian lecithin:cholesterol acyltransferase (LCAT) deficiency families have been investigated for mutations in the gene for lecithin:cholesterol acyltransferase by DNA sequencing of the exons amplified by the polymerase chain reaction. A single T→A transversion in codon 252 in exon 6 converting Met(ATG) to Lys(AAG) was observed in all homozygotes. In spite of the identical mutation, the disease phenotypes differed in severity. This was not reflected in the expression of LCAT in the heterozygotes.
Cell type‐specific tyrosine phosphorylation of IL‐2 receptor β chain in response to IL‐2
Authors: Kumaki Satoru , Asao Hironobu , Takeshita Toshikazu , Kurahayashi Yumiyo , Nakamura Masataka , Beckers Thomas , Engels Joachim W. , Sugamura Kazuo
Published: July 2002

Functional activities of the IL‐2 receptor (IL‐2R) β chain exogenously expressed on lymphoid and non‐lymphoid cells were examined in terms of phosphorylation of IL‐2Rβ and cell growth. Lymphoid MOLT‐4 and its transfectants expressing IL‐2Rβ either alone or with IL‐2R α chain were found to be rapidly phosphorylated predominantly at tyrosine residues of IL‐2Rβ and to be affected in their growth in an IL‐2‐dependent manner. In contrast, IL‐2 induced neither phosphorylation of IL‐2Rβ nor cell growth in non‐lymphoid transfectants derived from COS7, HeLa and L929, even though they acquired the IL‐2 binding ability when coexpressed as IL‐2Rβ and IL‐2Rα. These results suggest that IL‐2 induces activation of a tyrosine kinase possibly associated with IL‐2Rβ in a cell type‐specific manner.
Photolabeling of the phosphate binding site of chloroplast coupling factor 1 With [32P]azidonitrophenyl phosphate
Authors: Michel Laurent , Garin Jérôme , Girault Guy , Vignais Pierre V.
Published: July 2002

Chloroplast F1‐AT Pase (CF1) was photolabeled by a radiolabeled photoactivatable derivative of Pi, 4‐azido‐2‐nitrophenyl [32P]phosphate (ANPP). The radioactivity was localized in the β subunit of CF1. Upon cleavage of the β subunit by cyanogen bromide, the predominantly labeled peptide was recovered, which was subsequently subjected to tryptic digestion. A tryptic peptide (spanning Ile312‐Arg354), was found to contain nearly all the covalently bound radioactivity. By Edman degradation, the labeled amino acid residues were identified as Tyr328, Val329 and Pro330. The labeled β‐Tyr328 of CF1 is the equivalent of β‐Tyr311 of F1 from beef heart mitochondria, which was previously found to be photolabeled by ANPP [J. Garin et al. (1989) Biochemistry 28, 1442–1448].