bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of 2‐chloroadenosine on the nucleotide content of isolated rat hepatocytes
Authors: Claeyssens Sophie , Hamet Michèle , Chedeville Arlette , Basuyau Jean-Pierre , Lavoinne Alain
Published: July 2002

2‐Chloroadenosine is presumably a non‐metabolizable analogue of adenosine; however, this compound induced an increase in the enzymatically measured nucleotide content of isolated rat hepatocytes. HPLC separation and spectral analysis of the peaks showed that this increase may be related to the formation of 2‐chloro nucleotides and that the 2‐chloro nucleotides appeared in the first minutes of the incubation period. These results demonstrate that 2‐chloroadenosine may be metabolized by phosphorylation in rat liver cells.
Phosphoinositide breakdown and superoxide anion release in formyl‐peptide‐stimulated human alveolar macrophages Comparison between quiescent and activated cells
Authors: Damon Marcelle , Vial Henri , de Paulet André Crastes , Godard Philippe
Published: July 2002

Human alveolar macrophages (AMs) from allergic asthmatics (AAs) showed continuous Li+‐sensitive production of IP1, indicating that the cells were continuously activated. Furthermore, whereas the accumulation of IP1, IP2 and IP3 rapidly increased by as much as 125—175% in chemotactic‐factor‐stimulated AMs from healthy subjects, stimulation of cells from AAs increased these inositol phosphates only slightly. This moderate production could be due to a permanent state of activation leading to a depleted pool of polyphosphoinositides, corroborating the greater capacity of these cells to generate superoxide anion after stimulation by a chemoattractant. The activation state could be due to the action of priming agents, which are known to be released into the inflammatory sites.
UV‐induced cross‐linking of proteins to plasmid pBR322 containing 8‐azidoadenine 2′‐deoxyribonucleotides
Authors: Meffert Rainer , Dose Klaus
Published: July 2002

An efficient method of cross‐linking DNA to protein is described. The method is based on the incorporation of photoactive 8‐azidoadenine 2′‐deoxyribonucleotides into DNA. We have found that 8‐N3dATP is a substrate for E. coli DNA polymerase I and that 8‐N3dATP can be incorporated into plasmid pBR322 by nick‐translation. Subsequently we were able to cross‐link a set of different proteins to 8‐azido‐2′‐deoxyadenosine‐containing pBR322 by UV irradiation (366 nm). No DNA‐protein photocross‐linking was observed under the same conditions when the non‐photoactive plasmid pBR322 was used.
A cAMP‐triggered release of a hormone‐like peptide
Authors: Riven-Kreitman Rivka , Ribon Vered , Tauber-Finkelstein Miriam , Shaltiel Shmuel
Published: July 2002

Preparations of the catalytic subunit of cAMP‐dependent protein kinase from rabbit skeletal muscle, which appear to be homogeneous by SDS‐polyacrylamide gel electrophoresis, were often found to contain a hormone‐like factor (HLF) which causes an immediate rise, then a decline of intracellular cAMP in a B‐lymphoma cell line. Active HLF is released when the fractions that contain it in an inactive form are incubated with cAMP prior to chromatography, or passed through an immobilized cAMP column. HLF seems to be a peptide: it loses its cell‐stimulating capability after proteolysis and has an apparent molecular mass of 2.2 – 2.5 kDa.
Regulation of polypeptide‐chain initiation in rat skeletal muscle Starvation does not alter the activity or phosphorylation state of initiation factor eIF‐2
Authors: Cox Sarah , Redpath Nicholas T. , Proud Christopher G.
Published: July 2002

In rats, 48‐h starvation causes a decrease in the rate of protein synthesis in skeletal (e.g. gastrocnemius) muscle, due largely to impairment of peptide‐chain initiation. In other cell types inhibition of initiation is associated with decreased activity and recycling of initiation factor eIF‐2, and increased phosphorylation of its α‐subunit. However, 48‐h starvation has no effect on the activity or recycling of eIF‐2 measured in extracts of gastrocnemius muscle, or on the level of α‐subunit phosphorylation. The effects of starvation on peptide‐chain initiation in skeletal muscle must therefore involve alterations in other components of the translational machinery.
Irreversible inactivation of Saccharomyces cerevisiae fructose‐1,6‐bisphosphatase independent of protein phosphorylation at Ser11
Authors: Rose Matthias , Entian Karl-Dieter , Hofmann Lucia , Vogel Rudi F. , Mecke Dieter
Published: July 2002

The fructose‐1,6‐bisphosphatase gene was used with multicopy plasmids to study rapid reversible and irreversible inactivation after addition of glucose to derepressed Saccharomyces cerevisiae cells. Both inactivation systems could inactivate the enzyme, even if 20‐fold over‐expressed. The putative serine residue, at which fructose‐1,6‐bisphosphatase is phosphorylated, was changed to an alanine residue without notably affecting the catalytic activity. No rapid reversible inactivation was observed with the mutated enzyme. Nonetheless, the modified enzyme was still irreversibly inactivated, clearly demonstrating that phosphorylation is an independent regulatory circuit that reduces fructose‐1,6‐bisphosphatase activity within seconds. Furthermore, irreversible glucose inactivation was not triggered by phosphorylation of the enzyme.
Identification of two distinct protein carboxyl methyltransferases in eucaryotic cells
Authors: Solomon Roya , Katzir Orit , Egozi Yaakov , Kloog Yoel
Published: July 2002

Two distinct protein carboxyl methyltransferases (PCM) were identified in the electric organ of Torpedo ocellata. They were separated from each other in the active form by means of nondenaturing gel electrophoresis and by p‐(chloromercuri) benzoate‐agarose chromatography, and were individually identified by specific polyclonal antibodies. The existence of at least two distinct PCMs in eucaryotic cells raises the possibility that these enzymes are involved in distinct transmethylation reactions.
Identification of EcoRV fragments spanning the Nα‐tubulin gene of Physarum
Authors: Watts David I. , Monteiro Mervyn J. , Cox Robert A.
Published: July 2002

The Nα‐tubulin gene of Physarum polycephalum has an EcoRV site at codons 252/253. EcoRV digestion of Physarum DNA generated two EcoRV fragments per gene copy comprising both coding and flanking sequences. Hybridisation probes which included coding sequences upstream from the central EcoRV site cross‐hybridised with another α‐tubulin gene. Probes derived from either 5′‐ or 3′‐flanking regions were gene‐specific. These probes identified two EcoRV fragments in the haploid strain CLdAXE viz 5.4 kb (5′‐fragment) and 6.2 kb (3′‐fragment). The same two fragments were identified in EcoRV digests of DNA of the diploid strain M3CVIII, and a second form of the gene was also identified comprising two fragments viz 5.0 kb (5′‐end) and 5.5 kb (3′‐end). Both forms gave rise to an identical 4.65 kb HindIII fragment as judged by restriction mapping.
Evidence of a yeast proteinase specific for elongation factor 2
Authors: Servillo L. , Quagliuolo L. , Balestrieri C. , Giovane A.
Published: July 2002

Two proteinases active on elongation factor 2 have been found in yeast. The former hydrolyzes the factor producing a single ADP‐ribosylatable fragment, whereas it does not produce any fragment when incubated with different proteins. The latter, less specific, is active in cleaving both EF‐2 and other proteins giving rise to a noticeable number of fragments. Moreover, when native EF‐2 is incubated with the most specific of the two proteinases, the amount of the ADP‐ribosylatable fragment increases with time, while no fragments are evident when ADP‐ribosylation of EF‐2 comes before its incubation with the proteolytic enzyme. A possible regulatory role of this proteinase on EF‐2 turnover is hypothesized.
Phospholipid flippases
Authors: Devaux Philippe F.
Published: July 2002

Protein mediated phospholipid translocation through membranes has been observed in rat liver endoplasmic reticulum and in the plasma membrane of erythrocytes as well as in a few other cell membranes. Lipid translocation in plasma membranes is ATP dependent and selectively accumulates aminophospholipids on the inner monolayers.
Structural specificity of haemosiderin iron cores in iron‐overload diseases
Authors: Mann S. , Wade V.J. , Dickson D.P.E. , Reid N.M.K. , Ward R.J. , O'Connell M. , Peters T.J.
Published: July 2002

Haemosiderin iron cores isolated from patients with secondary haemochromatosis have a goethite‐like (α‐FeOOH) crystal structure whereas those from patients with primary haemochromatosis are amorphous Fe (III) oxide. Haemosiderin cores isolated from normal human spleen are crystalline ferrihydrite (5Fe2O3·9H2O). The disease‐specific structures are significantly different from the ferrihydrite structure of associated ferritin cores. The results are important in understanding the biological processing of iron in pathological states and in the clinical treatment of iron‐overload diseases.
Immunological studies on the D‐1 and D‐2 proteins of photosystem II preparations from the thermophilic cyanobacterium, Synechococcus sp
Authors: Satoh Kazuhiko , Dostatni Ralf , Johanningmeier Udo , Oettmeier Walter
Published: July 2002

Immunoblotting of Synechococcus photosystem II preparations with site‐directed antisera against the spinach D‐2 protein and an antiserum against a large polypeptide segment of the spinach D‐1 protein showed (i) that the 28 and 31 kDa polypeptides of the cyanobacterial photosystem II core complex are equivalent to the D‐1 and D‐2 proteins of higher plants and algae and (ii) that the β‐octylglucoside treatment of the photosystem II preparation diminishes band intensities of the 28 and 31 kDa proteins in Coomassie blue‐stained gels mainly by increasing the amounts of two proteins which migrate as a dimer of D1 or D2 or its aggregate, and of proteolytic digestion products.
Epidermal growth factor‐induced increases in inositol trisphosphates, inositol tetrakisphosphates, and cytosolic Ca2+ in a human hepatocellular carcinoma‐derived cell line
Authors: Gilligan Ann , Prentki Marc , Glennon Major C. , Knowles Barbara B.
Published: July 2002

A human hepatocellular carcinoma‐derived cell line, PLC/PRF/5, was examined for its ability to respond to epidermal growth factor (EGF) exposure with increased phosphatidylinositol 4,5‐bisphosphate hydrolysis. Upon addition of EGF (25 ng/ml) a rapid (10–15 s) but transient increase in Ins(1,4,5)P3 levels and large, prolonged (2 min) increases in Ins(1,3,4,5)P4 andIns(1,3,4)P3 levels were detected. Increases in cytosolic Ca2+ were observed after a 10 to 2O s lag, reaching peak value at 1 min, and remaining elevated for 10 min. The initial burst of cytosolic Ca2+ occurred in the absence of extracellular Ca2+ and probably reflects mobilization of intracellular Ca2+ stores. In cells pretreated with EGTA, the sustained component of the Ca2+ response was not observed. Comparison of the inositol phosphate and Ca2+ responses of PLC/PRF/5 cells to responses reported in other cell types indicates that this cell line is a good model for EGF action in liver.
The relationship between superoxide generation, cytochrome b and oxygen in activated neutrophils
Authors: Edwards Steven W. , Lloyd David
Published: July 2002

The O2 affinity for O.
2 — generation (i.e. O2 tension at which 50% of the O2‐saturated activity was observed) by rat neutrophils activated by the chemotactic peptide (N‐formyl‐methionyl‐leucyl‐phenylalanine) plus cytochalasin B was measured as 5.4 μM (± 1.4, n = 5). Similarly, the O2 tension at which cytochrome b was oxidized to 50% of its aerobic steady‐state level in activated cells was 4.7 μM (± 1.0, n = 3): in non‐activated cells the corresponding value for cytochrome b oxidation was 11.4 μM (± 2.8, n = 3). It is proposed that O2 depletion at inflammatory sites may limit oxidant generation by activated neutrophils and thus reduce oxidant damage to surrounding tissues.
A protein of Halobacterium halobium immunologically related to the v‐myc gene product
Authors: Ben-Mahrez Kamel , Perbal Bernard , Kryceve-Martinerie Cecile , Thierry Dominique , Kohiyama Masamichi
Published: July 2002

A 70 kDa protein of Halobacterium halobium cross‐reacts with an antiserum directed against the v‐myc gene product of the avian myelocytomatosis virus (MC29). This cross‐reaction is in agreement with hybridization studies which indicate that H. halobium possesses DNA and RNA sequences homologous to the v‐myc gene.
Separation of the pore‐forming and cytotoxic activities from natural killer cell cytotoxic factor
Authors: Sashchenko L.P. , Gnuchev N.V. , Kirillova M.A. , Lukjanova T.I. , Rebizova T.V. , Revazova E.S. , Lukanidin E.M.
Published: July 2002

The influence of Ca2+ on the cytotoxic activity of natural killer cell cytotoxic factor (NKCF) was analyzed. When the natural killer susceptible cell line K562 was exposed to NKCF in the presence of 5 mM Ca2+, two peaks of cell damage were found. The first peak was observed after 30–40 min of incubation as a result of pore formation on the surface of target cells. The second was a peak of cytolytic activity which appeared after 24 h of incubation. Upon dilution of the NKCF preparation, only the first peak was observed. Therefore, NKCF produced by large granular lymphocytes in response to K562 consists of different proteins and represents pore‐forming and cytolytic activities.
Induction of c‐fos protein by activation of vasopressin receptors in smooth muscle cells
Authors: Nambi P. , Watt R. , Whitman M. , Aiyar N. , Moore J.P. , Evan G.I. , Crooke S.
Published: July 2002

Stimulation of vasopressin (V1) receptors of rat aortic smooth muscle cells (A‐10, ATCC CRL 1476) results in the hydrolysis of phosphatidylinositol 4,5‐bisphosphate (PIP2) to inositol 1,4,5‐trisphosphate (IP3) and diacylglycerol (DAG) with the mobilization of intracellular calcium. When A‐10 cells are exposed to arginine vasopressin (AVP), there is an increase in the level of c‐fos oncoprotein. The extent of induction of c‐fos oncoprotein depends on both the time of exposure of the cells to AVP, reaching a maximum at 60 min after which there is a slow decline, and the concentration of AVP used, with an approximate EC50 of 1 nM which corresponds well with the Kd of vasopressin binding to these receptors. This vasopressin‐mediated increase in c‐fos protein level is inhibited by a V1/V2 antagonist (SKF 101498) suggesting that this is a receptor‐mediated event. In addition dDAVP, a V2 selective agonist, is much less effective than AVP in inducing c‐fos protein suggesting that AVP mediates its effect via V1 receptors. Desensitization of vasopressin receptors by prolonged exposure to AVP resulted in no additional induction of c‐fos protein level in response to second challenge of AVP. In addition to AVP, phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC), also stimulates the accumulation of c‐fos protein although to a lesser extent than AVP. The above data suggest that c‐fos protein levels in smooth muscle cells are regulated by AVP and the hormonal effect may be mediated through PI turnover and DAG, IP3 and Ca2+ signals.
Mode of binding of E‐64‐c, a potent thiol protease inhibitor, to papain as determined by X‐ray crystal analysis of the complex
Authors: Matsumoto Keita , Yamamoto Daisuke , Ohishi Hirofumi , Tomoo Kouji , Ishida Toshimasa , Inoue Masatoshi , Sadatome Takahisa , Kitamura Kunihiro , Mizuno Hiroshi
Published: July 2002

The three‐dimensional structure of the E‐64‐c‐papain complex has been determined by X‐ray crystal analysis at 2.5 Å resolution (conventional R = 26.9%). The structure determined indicates that: (i) the C2 atom of the oxirane ring of E‐64‐c is covalently bound by the Sγ atom of Cys‐25 of papain; (ii) this covalent bond formation results in a configurational conversion of the oxirane C2 atom from the S‐ to the R‐form; and (iii) extensive hydrogen bonding and hydrophobic interactions are responsible for the specific interaction of the E‐64‐c molecule with papain.
The complete amino acid sequence of ribosomal protein S18 from the moderate thermophile Bacillus stearothermophilus
Authors: McDougall John , Choli Theodora , Kruft Volker , Kapp Ulrike , Wittmann-Liebold Brigitte
Published: July 2002

The amino acid sequence of ribosomal protein S18 from Bacillus stearothermophilus has been completely determined by automated sequence analysis of the intact protein as well as of peptides derived from digestion with Staphylococcus aureus protease at pH 4.0 and cleavage with cyanogen bromide. The carboxy‐terminal region was verified by both amino acid analyses of chymotryptic peptides and by mass spectrometry from the terminal region. The protein contains 77 amino acid residues and has an M
r of 8838. Comparison of this sequence with the sequences of the S18 proteins from tobacco and liverwort chloroplasts and E. coli shows a relatively high similarity, ranging from 42 to 55% identical residues with the B. stearothermophilus S18 protein. The regions of homology common to all four proteins consist of several positively charged sections spanning the entire length of the protein.
Evidence that the anti‐coagulant and lethal properties of a basic phospholipase A2 from snake venom are unrelated
Authors: Chwetzoff Serge , Couderc Jacques , Frachon Paule , Menez André
Published: July 2002

The basic phospholipase A2 (PLA2) from venom of the African elapid Naja nigricollis was previously shown to have anti‐coagulant and lethal properties, both of which were abolished by treatment with p‐bromophenacyl bromide (pBP). In the present paper we first report that pBP‐treated PLA2 is capable of inhibiting the anti‐coagulant activity but not the lethal activity of native PLA2, thus suggesting that both properties might be independent. We then confirm this evidence using PLA2‐specific monoclonal immunoglobulins. One of these, called HSF, neutralized the lethal activity but not the anti‐coagulant activity, whereas another antibody, called HSP2, inhibited the anti‐coagulant activity but not the lethal activity of the PLA2. The data presented in this paper are taken as evidence that the anti‐coagulant activity is not implicated in the lethal effects of basic PLA2 from Naja nigricollis.
O‐Mannosylation of recombinant human insulin‐like growth factor I (IGF‐I) produced in Saccharomyces cerevisiae
Authors: Hård Karl , Bitter Wilbert , Kamerling Johannis P. , Vliegenthart Johannes F.G.
Published: July 2002

A glycosylated form of recombinant human insulin‐like growth factor I (IGF‐I) expressed in Saccharomyces cerevisiae was shown to contain mannose as the only carbohydrate constituent. All oligosaccharide chains of the glycoprotein could be released by mild alkaline treatment, and separated from the protein by gel‐permeation chromatography on Bio‐Gel P‐2. The structures of these O‐linked carbohydrate chains were determined by 500‐MHz 1H‐NMR spectroscopy, affording the disaccharide Manα1‐2Man as the major component and the tetrasaccharide Manα1‐3Manα1‐2Manα1‐2Man as a minor component. Reference oligosaccharides were prepared from mannoproteins released from the cell wall of S. cerevisiae X2180 (α‐wild type). In addition to previously reported structures, ranging from mannose to mannotetraose, the pentasaccharide Manα1‐3Manα1‐3Manα1‐2Manα1‐2Man was identified in the cell wall mannoprotein.
Mitochondrial metabolism of guanine nucleotides possible role of guanosine
Authors: Ziegler Mathias , Dubiel Wolfgang , Pimenov Alexander M. , Tikhonov Yuri V. , Toguzov Rusian T. , Henke Wolfgang , Gerber Gerhard
Published: July 2002

The catabolism of intramitochondrial guanine nucleotides was examined. During 30 min incubation of rat liver mitochondria at 37°C in the presence of oligomycin and carboxyatractyloside, guanine and xanthine were formed and appeared in the medium. Under these conditions, the direct conversion of GMP to guanine by hypoxanthine—guanine phosphoribosyltransferase is suggested to be the main catabolic route within the organelles. Only very small amounts of guanosine were produced and detected both inside and outside the organelles. [14C]Guanosine and [14C]inosine were taken up by the mitochondria. Therefore, guanosine is suggested to be a precursor of intramitochondrial guanine nucleotides.
Interaction of benz[a]pyrene diol epoxide with chromatin studied by flow linear dichroism
Authors: Eriksson S. , Jernström B. , Nielsen P.E. , Nordén B.
Published: July 2002

Chromatin isolated from Ehrlich ascites cells was incubated with the tumourigenic compound (+)‐7β,8α‐dihydroxy‐9α,10α‐epoxy‐7,8,9,10‐tetrahydrobenz[a]pyrene [(+)‐anti‐BPDE] at low ionic strength and the modified chromatin was analysed using flow linear dichroism (LD). The results confirm that (+)‐anti‐BPDE preferentially binds to the DNA in the linker regions, and furthermore show that the long axis of the bound pyrenyl chromophore is oriented parallel or close to parallel to the average orientation of the chromatin fiber axis. The data indicate that the binding geometry of (+)‐anti‐BPDE in chromatin is similar to that in pure DNA and deoxyguanosine‐containing double‐helical oligonucleotides.
Retrotransposon transposition intermediates are encapsidated into virus‐like particles
Authors: Gorelova T.V. , Resnick N.L. , Schuppe N.G.
Published: July 2002

Virus‐like particles (VLPs) possessing reverse transcriptase activity are persistently present in Drosophila melanogaster cultured cells and are formed in yeast induced for transposition. Different retrotransposon transposition intermediates consistent with those expected from the model of reverse transcription pathway of retrotransposon transposition have been detected during the analysis of nucleic acids isolated from VLPs. These data indicate that the act of reverse transcription takes place in VLPs which may be considered as functional intermediates of transposition.
Identification of the bovine brain Ins(1,4,5)P3 5‐phosphatase after SDS‐polyacrylamide gel electrophoresis
Authors: Lemos Manuela , Dumont Jacques E. , Erneux Christophe
Published: July 2002

Ins(1,4,5)P3 5‐phosphatase catalyses the dephosphorylation of Ins(1,4,5)P3 in the 5‐position. In a high speed soluble fraction of bovine brain, there are two soluble 5‐phosphatases: type I and type II. The purified Ins(1,4,5)P3 5‐phosphatase type I exhibits a major silver‐stained band of 43 kDa on denaturing (SDS) gels. It is possible to extract the 5‐phosphatase activity form a duplicate lane after gel electrophoresis. The 43 kDa region contains the extractable Ins(1,4,5)P3 5‐phosphatase activity.
Mitochondrial F0F1 H+‐ATP synthase Characterization of F0 components involved in H+ translocation
Authors: Guerrieri Ferruccio , Capozza Giuseppe , Houštěk Josef , Zanotti Franco , Colaianni Gina , Jirillo Emilio , Papa Sergio
Published: July 2002

The membrane F0, sector of mitochondrial ATP synthase complex was rapidly isolated by direct extraction with CHAPS from F1‐depleted submitochondrial particles. The preparation thus obtained is stable and can be reconstituted in artificial phospholipid membranes to result in oligomycin‐sensitive proton conduction, or recombined with purified F1 to give the oligomycin‐sensitive F0F1‐ATPase complex. The F0 preparation and constituent polypeptides were characterized by SDS‐polyacrylamide gel electrophoresis and immunoblot analysis. The functional role of F0 polypeptides was examined by means of trypsin digestion and reconstitution studies. It is shown that, in addition to the 8 kDa DCCD‐binding protein, the nuclear encoded protein [(1987) J. Mol. Biol. 197, 89–100], characterized as an intrinsic component of F0, (F0I, PVP protein [(1967) J. Biol. Chem. 242, 2547–2551]) is involved in H+ translocation and the sensitivity of this process to the F0 inhibitors, DCCD and oligomycin.
The primary structure of Pseudomonas cytochrome c peroxidase
Authors: Rönnberg Marjaana , Kalkkinen Nisse , Ellfolk Nils
Published: July 2002

The primary structure of Pseudomonas cytochrome c peroxidase is presented. The intact protein was fragmented with cyanogen bromide into five fragments; partial cleavage was observed at a Met‐His bond of the protein. The primary structure was established partly by automatic Edman degradations, partly by manual sequencing of peptides obtained with trypsin, thermolysin, chymotrypsin, pepsin, subtilisin and Staphylococcus aureus V8 endopeptidase. The order of the cyanogen bromide fragments was further confirmed by overlapping peptides obtained by specific cleavage of the whole protein. Pseudomonas cytochrome c peroxidase consists of 302 amino acid residues giving a calculated M
r of 33 690.
Identification of a wheat germ agglutinin‐sensitive ATPase in yeast nuclei
Authors: Tsuchiya Eiko , Hiraga Kazumi , Fukui Sakuzo , Miyakawa Tokichi
Published: July 2002

We have found that wheat germ agglutinin (WGA), a lectin that specifically binds to N‐acetylglucosamine residues inhibits the in vitro transport of plasmid DNA, pJDB219, into yeast nuclei. Histochemical staining of the isolated nuclei with biotinylated WGA and streptavidin‐biotinylated peroxidase complex revealed the presence of WGA‐binding materials around the nuclear pore under an electron microscope. Using WGA‐agarose column chromatography of yeast nuclear extracts, a novel Mg2+‐dependent ATPase was isolated. Its activity was highly sensitive to WGA and stimulated by Nonidet P‐40 or phosphatidylserine. We suggest that the WGA‐sensitive ATPase plays a role in yeast nuclear transport of DNA.
The effect of the replacement of ADP with a photoaffinity ATP analogue, 2‐azido‐ADP, in F‐actin on its function
Authors: Kuwayama Hideto , Miki Masao , dos Remedios Cristobal G.
Published: July 2002

2‐Azido‐ATP, a photoaffinity ATP analogue, was incorporated into actin and the influence of the incorporation on the actin function was studied. The replacement of ADP with 2‐azido‐ADP in F‐actin both before and after photocross‐linking decreased appreciably the actin‐activated S1‐ATPase activity. Photocross‐linked 2‐azido‐ADP‐F‐actin could be depolymerized by dialysis against a solution containing 0.1 mM CaCl2, 0.1 mM ATP and 1 mM Tris‐HCl (pH 8.0). However, once it depolymerized, it lost very quickly the ability to polymerize even in the presence of a sufficient amount of ATP and Ca2+.
Protein kinase C is crucial in signal transduction during IFN‐γ induction in endothelial cells
Authors: Mattila Pirkko , Häyry Pekka , Renkonen Risto
Published: July 2002

We have demonstrated that IFN‐γ, a potent peptide mediator in inflammatory responses, operates via the protein kinase C dependent transduction pathway in the induction of class II MHC antigens on rat microvascular endothelial cells. Stimulators of protein kinase C, like PMA, replaced IFN‐γ in the induction of MHC class II on endothelial cells in a dose‐dependent manner. Selective enzyme inhibitors of protein kinase C, H‐7 as well as sphingosine down‐regulated the IFN‐γ induced class II expression in a dose‐dependent manner. Addition of cAMP or cGMP in the culture, had no effect on the class II expression on the endothelial cells. Transient rise of cytosolic Ca2+ by calcium ionophore A23187, or a calmodulin antagonist W‐7, had no effect on the IFN‐γ induced class II expression.
Concanavalin A stimulation of O2 consumption in electropermeabilized neutrophils via a pertussis toxin‐insensitive G protein
Authors: Lu Diane J. , Grinstein Sergio
Published: July 2002

Electropermeabilized human neutrophils were used to investigate the possible role of G‐proteins in the respiratory burst elicited by concanavalin A (Con A). The Con A‐induced activation of the NADPH oxidase was not inhibited by either pertussis toxin or cholera toxin. However, the burst was inhibited by GDP and GDPβS providing evidence for the involvement of a G‐protein(s). O2 consumption in Con A‐stimulated cells was dependent on both ATP and Mg2+. ATP could be substituted by ATPγS but not by the non‐hydrolyzable analog AMP‐PNP, suggesting involvement of phosphotransferase reactions. It is concluded that at least two distinct types of G‐proteins are capable of inducing the respiratory burst in neutrophils and that accumulation of phosphorylated intermediates may be essential for activation of the respiratory burst by the lectin.
Purification of the mitochondrial carnitine carrier by chromatography on hydroxyapatite and celite
Authors: Indiveri C. , Palmieri F.
Published: July 2002

The carnitine carrier from rat liver mitochondria has been extracted with Triton X‐100 and partially purified by chromatography on hydroxyapatite and celite. During purification the activity of the carrier was monitored by functional reconstitution into liposomes. The purified fraction is 250‐fold enriched with respect to the N‐ethylmaleimide‐sensitive carnitine/carnitine transport activity. The substrate specificity and the inhibitor sensitivity of carnitine transport in liposomes resemble closely those described for the transport of carnitine in mitochondria.
Probing the nucleotide‐binding site of sarcoplasmic reticulum (Ca2+‐Mg2+)‐ATPase with anti‐fluorescein antibodies
Authors: Mata A.M. , Lee A.G. , East J.M.
Published: July 2002

Antibodies raised against fluorescein were unable to bind to the fluorophore when bound at the nucleotide‐binding site of native (Ca2+‐Mg2+)‐ATPase, as judged by fluorescence quenching assays or competitive ELISAs, but were able to bind when the ATPase was denatured. Indirect ELISAs, in which native and denatured FITC‐ATPase were used to coat ELISA plates, were unable to detect the difference in accessibility of the fluorescein bound to the native and denatured ATPase. These results indicate that the nucleotide‐binding site is relatively inaccessible in the native structure, even though fluorescence energy transfer studies [(1987) Biochim. Biophys. Acta 897, 207–216] indicate that this site must be close to the surface of the ATPase. In addition the results suggest that the indirect ELISA method may be of limited value in probing the accessibility of epitopes using antibodies.
Enhancement of arachidonic acid liberation by protein kinase C activator is partially dependent on extracellular Na+ in rabbit platelets
Authors: Akibaaf Satoshi , Sato Takashi , Fujii Tatsuzo
Published: July 2002

In [3H]arachidonic acid‐labeled rabbit platelets, pretreatment with phorbol 12‐myristate 13‐acetate (20 nM) or dioctanoylglycerol (20 μM) enhanced [3H]arachidonic acid liberation induced by low concentration of A23187 (150 nM). When extracellular Na2+ was replaced with N‐methyl‐D‐glucamine, the enhancement is reduced by about 50%. Similar synergistic enhancement of the liberation was obtained by using monensin (2–10 μM) or NH4Cl (5–20 mM) in place of protein kinase C activator in combination with A23187. The guanosine 5′‐O‐[3‐thiotriphosphate] (100 μM)‐induced liberation was also enhanced by a rise of extracellular pH (pH 7.0–7.8) in saponin‐permeabilized platelets. These results suggest that the enhancement of arachidonic acid liberation by protein kinase C may partially be mediated by intracellular alkalinization in rabbit platelets.
Direct observation of substrate binding to ferrous‐CO cytochrome P‐450‐CAM using 19F NMR
Authors: Crull George B. , Nardo Joseph V. , Dawson John H.
Published: July 2002

The binding of two fluorinated substrate analogs, 9‐fluorocamphor and 5,5‐difluorocamphor, to cytochrome P‐450‐CAM has been investigated by 19F NMR spectroscopy. The NMR properties of each substrate differ depending on whether it is free in aqueous buffer, bound to the diamagnetic ferrous‐CO enzyme or bound to the paramagnetic ferrous derivative. As CO must be bound to the ferrous center for it to be diamagnetic, these results demonstrate that camphor and CO bind to the protein simultaneously. The present results are unusual in that the spectral properties of the substrate rather than those of the heme iron have been monitored to follow substrate and ligand binding.
Amino acid sequence around the thiolester of α2‐macroglobulin from plasma of the crayfish, Pacifastacus leniusculus
Authors: Hall Martin , Söderhäll Kenneth , Sottrup-Jensen Lars
Published: July 2002

α2‐Macroglobulin (α2M) was isolated from plasma of the freshwater crayfish, Pacifastacus leniusculus, using ultracentrifugation, ion‐exchange chromatography and gel filtration techniques. The Pacifastacus α2M molecule (Pα2M) was radioactively labeled in the thiol ester structure with iodo [14C]acetic acid in the presence of methylamine. After reduction and carboxymethylation of the protein, it was digested with trypsin. A 14C‐labeled tryptic peptide was sequenced and contained an amino acid sequence very similar to other known thiol ester sequences from human α2M and related proteins. The N‐terminal sequence of Pα2M was related to that recently determined for lobster α2M [(1987) J. Biol. Chem. 262, 14606–14611].
Flux‐yield dependence of oxidative phosphorylation at constant ΔH+
Authors: Ouhabi Rachid , Rigoulet Michel , Guerin Bernard
Published: July 2002

By using an NADH‐regenerating system, different steady states were obtained in which the electron flux through each respiratory unit was modulated. Under these conditions it was observed that, at constant ΔH+, the ATP/O value decreased with increasing electron flux. This flux‐yield dependence can be regarded as evidence in support of indirect coupling at the proton pump level.
Photoaffinity labeling of thiamin‐binding component in yeast plasma membrane with [3H]4‐azido‐2‐nitrobenzoylthiamin
Authors: Nishimura Hiroshi , Sempuku Kenji , Kawasaki Yuko , Nosaka Kazuto , Iwashima Akio
Published: July 2002

When prepared from Saccharomyces cerevisiae through an acid precipitation at pH 5.0 for a crude particulate fraction obtained by mechanical agitation of yeast protoplasts with glass beads, the plasma membranes have more remarkable binding quantities of [14C]thiamin (K
d, 51 nM; B
max, 263 pmol per mg of protein) compared with our previously prepared membranes [(1986) Experientia 42, 607–608]. Photoaffinity labeling of these yeast plasma membranes with [3H]4‐azido‐2‐nitrobenzoylthiamin resulted in the covalent modification of a membrane component with an apparent molecular mass of 6–8 kDa. The extent of its labeling was markedly decreased by previous addition of thiamin. This result suggests that the small membrane component (6–8 kDa) takes part in the thiamin binding of thiamin carrier protein(s) in yeast plasma membranes.
De novo biosynthesis and base composition of total and ribosomal ribonucleic acids during early development in Artemia sp.
Authors: Liras Antonio , Cruz M.C.Hernández , Rotllán Pedro , Argomaniz Luisa , Llorente Pilar
Published: July 2002

De novo synthesis of total and ribosomal ribonucleic acids has been studied during the early stages of Artemia sp. development. By in vivo incorporation studies of [14C]HCO−
3 an increase has been found in both total and ribosomal RNA synthesis post hatching, with a similar distribution of radioactivity and base composition.
Irreversible binding of bacteriophage T5 to its FhuA receptor protein is associated with covalent cross‐linking of 3 copies of tail protein pb4
Authors: Feucht Andrea , Heinzelmann Gerrit , Heller Knut J.
Published: July 2002

Irreversible binding of bacteriophage T5 to its FhuA receptor protein is characterized by a high activation energy, typical for reactions where covalent bonds are formed [Zarnitz, M.L. and Weidel, W. (1963) Z. Naturforsch. 18b, 276–280]. Upon binding of radiolabeled T5 phages to FhuA formation of a new protein of 250 kDa was observed. Using electrophoretical and Western blotting techniques this protein was shown to be formed by cross‐linking of 3 copies of tail protein pb4, rather than by cross‐linking of FhuA and the receptor‐binding protein.
Electron donation by the high‐potential haems in Rhodopseudomonas viridis reaction centres at low temperatures
Authors: Hubbard J.A.M. , Evans M.C.W.
Published: July 2002

EPR signals due to c‐type cytochromes in Rhodopseudomonas viridis reaction centres are reported. Haems with E
m = 380 mV (1), 310 mV (1) and about 0 mV (2) were identified. At redox potentials where the low‐potential cytochromes are oxidised, but the high‐potential cytochromes are reduced, photooxidation of the high‐potential cytochromes is observed as QA is photoreduced by low‐temperature (15 K) illumination. Cyt c‐556→cyt c‐559→reaction centre→QA However, the signal attributed to QA is only 30–40% of the intensity and is narrower than that observed when QA photoreduced with all the haems reduced. It is suggested that reduction of the low‐potential haems causes conformational changes in the reaction centre, altering the iron‐quinone interaction and the electron‐transfer process in the cytochrome chain.
Involvement of cytochrome bc1 complex and cytochrome c2 in the electron‐transfer pathway for NO reduction in a photodenitrifier, Rhodobacter sphaeroides f.s. denitrificans
Authors: Itoh Masahiko , Mizukami Sally , Matsuura Katsumi , Satoh Toshio
Published: July 2002

The electron‐transfer pathway for NO reduction in a photodenitrifier, Rhodobacter sphaeroides f.s. denitrificans, was studied. A sample of membrane proteins containing cytochrome bc
1 complex and NO reductase activity was prepared from chromatophores using cholate/deoxycholate as detergents. The NO reductase activity was separated from the cytochrome bc
1 complex by ion‐exchange chromatography in the presence of dodecyl maltoside. When duroquinol was used as an electron donor, NO was reduced in the bc
1‐NO reductase preparation supplemented with cytochrome c
2. The reduction was inhibited by antimycin and myxothiazol. These results indicate that the cytochrome bc
1 complex, cytochrome c
2 and membranous NO reductase are involved in the electron‐transfer pathway from quinol to NO in this photodenitrifier.
In vitro reconstitution of hnRNP particles
Authors: Schenkel Johannes , Kloetzel Peter M.
Published: July 2002

The assembly of hnRNP‐like particles was studied by in vitro reconstitution, UV‐crosslinking and CsCl‐equilibrium centrifugation. Using total nuclear protein and RNA extracts from HeLa cells for RNP reconstitution, RNP particles sedimenting with the same buoyant density of ϱ=1.4 g/cm3 as ‘native’ 40 S core hnRNPs were obtained. Under the stringent reconstitution conditions used, hnRNP complexes containing only the C1‐core hnRNP protein could be identified.
Nucleotide sequence of Suncus murinus immunoglobulin μ gene and comparison with mouse and human μ genes
Authors: Ishiguro Hiroshi , Ichihara Yoshikazu , Namikawa Takao , Nagatsu Toshiharu , Kurosawa Yoshikazu
Published: July 2002

Both primates and rodents apparently originated from insectivores and then evolved separately. We isolated the immunoglobulin μ gene from DNA of the insectivore Suncus murinus and determined its nucleotide sequence. The gene organization was CH1 exon (318 bp)‐intron (89 bp)‐CH2 exon (345 bp)‐intron (80 bp)‐CH3 exon (318 bp)‐intron (85 bp)‐CH4 exon (392 bp)‐coding sequence and 3′‐untranslated region. Comparison of nucleotide sequences of μ genes between mouse, human and Suncus murinus indicated that the evolutionary distance between human and mouse is equal to that between Suncus murinus and human, and that mouse is deviated further from Suncus murinus than the two other combinations. This conclusion was further supported by sequence comparison of non‐coding regions.
Evidence for the induction of cytochrome P‐452 in rat liver by Aroclor 1254, a commercial mixture of polychlorinated biphenyls
Authors: Borlakoglu J.T. , Edwards-Webb J.D. , Dils R.R. , Wilkins J.P.G. , Robertson L.W.
Published: July 2002

We have examined the ability of a commercial mixture of polychlorinated biphenyls (Aroclor 1254) to induce hepatic cytochrome P‐452‐linked enzyme activities in rat and pigeon liver five days after its intraperitoneal injection. The results provide evidence that, at the doses used, Aroclor 1254 induces cytochrome P‐452‐linked enzyme activities in rats, but not in pigeons. This inductive effect was previously regarded as being specific for hypolipidemic drugs and phthalate ester plasticisers.
Two structural types of mercury reductases and possible ways of their evolution
Authors: Bogdanova E.S. , Mindlin S.Z.
Published: July 2002

Structural differences have been revealed among mercury reductases of immunologically unrelated types from Gram‐positive bacteria: enzymes of one immunological type have a molecular mass of 62–69 kDa and seem to contain an N‐terminal extension of 5–15 kDa, which is easily cleaved by trypsin and chymotrypsin; enzymes of the other immunological type have a molecular mass of 52–57 kDa and are resistant to proteolysis. The existence of at least two different lines in the evolution of mercury reductases is surmised.
Evidence for leukotriene C4 transport mediated by an ATP‐dependent glutathione S‐conjugate carrier in rat heart and liver plasma membranes
Authors: Ishikawa Toshihisa , Kobayashi Kazuo , Sogame Yoshihisa , Hayashi Koichiro
Published: July 2002

Using rat heart sarcolemma and liver plasma membrane vesicles, it has been verified that the transport of leukotriene C4 (LTC4) across membranes is an ATP‐dependent process; the apparent K
m for LTC4 was 150 nM (heart sarcolemma) or 250 nM (liver plasma membrane). S‐(2,4‐dinitrophenyl)‐glutathione (DNP‐SG) inhibited LTC4 uptake into the vesicles dose‐dependently (I
50 = 25 (μM for both heart sarcolemma and liver plasma membrane vesicles). Mutual inhibition between LTC4 and DNP‐SG in uptake into the vesicles demonstrates that transport of LTC4 is mediated by an ATP‐dependent glutathione S‐conjugate carrier.
The histones of Caenorhabditis elegans: no evidence of stage‐specific isoforms
Authors: Vanfleteren Jacques R. , Van Bunt Stefaan M. , Van Beeumen Jozef J.
Published: July 2002

The nematode Caenorhabditis elegans expresses one species of H2A and one species of H4 molecules, at least two species of H1 (H1.1, H1.2), two species of H2B (H2B.1, H2B.2) and 2–4 species of H3 (H3.1 and H3.3 and an unassigned Ile/Leu microheterogeneity in H3). The study of their primary structures has been completed now and all of them, with the exception of the Ile/Leu microheterogeneity in H3, have been assigned to protein spots on two‐dimensional gels. One spot, previously designated H3.2, probably represents C‐terminally cleaved H3.1. The relative abundance of the isohistones was essentially the same when derived from either eggs, gravid adults or postreproductive, senescent worms. The degree of post‐translational modification, however, particularly acetylation of H2A, H2B and H3 histone species, was reduced at old age.
Imaging of cell membraneous and cytoskeletal structures with a scanning tunneling microscope
Authors: Ruppersberg Johann Peter , Hörber J.K.Heinrich , Gerber Christoph , Binnig Gert
Published: July 2002

The first observation of unstained cell membraneous structures by a scanning tunneling microscope is reported. An adhesive preparation method was used for imaging human medulloblastoma cells from the cell line TE 671 and oocytes from the clawed toad Xenopus laevis. The images show filaments, stacks of molecules and hilly structures. The possible identity of the filamentous structures is discussed, although the observed structures cannot yet be fully characterized. The work suggests possible future experiments on various biological structures in their natural environment.
Inhibition of the first phosphodiester bond formation catalyzed by Escherichia coli RNA polymerase in the presence of bovine seminal plasmin: promoter dependency
Authors: Gopal Vijaya , Chatterji Dipankar
Published: July 2002

Inhibition of the abortive initiation of transcription catalyzed by E. coli RNA polymerase has been studied here in the presence of bovine seminal plasmin. Seminal plasmin, which is known to be a stronger inhibitor than rifampicin binds at the same site as rifampicin to RNA polymerase. However, unlike rifampicin, seminal plasmin showed the inhibition of the formation of both the first and second phosphodiester bonds. We observed, in vitro, that the degree of inhibition of transcription was different at different promoters. Thus, the percent of inhibition of transcription initiation by seminal plasmin was much less at r‐RNA promoters in comparison to that at the early promoters of bacteriophage T7.
Cloning of gene cluster for sarcotoxin I, antibacterial proteins of Sarcophaga peregrina
Authors: Kanai Akio , Natori Shunji
Published: July 2002

A genomic clone of sarcotoxin I was isolated. This clone contained four genes of structurally related proteins belonging to the sarcotoxin I family present in tandem array. One of these genes was sequenced and found to be the sarcotoxin IB gene. This gene contained a single intron of 95 bases.
A G‐protein mediates secretagogue‐induced gap junctional channel closure in pancreatic acinar cells
Authors: Somogyi Roland , Kolb Hans-Albert
Published: July 2002

Using the double whole‐cell patch‐clamp technique, we determined that dialysis of cell pairs by GTP[S] potentiated electrical uncoupling induced by extracellular addition of carbamylcholine (CCh). An inhibitor of diglyceride lipase, RHC 80267, further potentiated CCh/GTP[S]‐induced junctional channel closure, probably by accumulation of diacylglycerol. Moreover, the protein kinase C inhibitor polymyxin B completely blocked uncoupling elicited by CCh/GTP[S]. These results provide the first evidence suggesting that gap junction channel closure by cholinergic stimulation is mediated by a G‐protein, which acts by increasing phosphatidylinositol biphosphate breakdown and protein kinase C activity.
Proteinic prorenin‐releasing‐stimulator (PRS) in the rat submandibular gland
Authors: Suzuki Fumiaki , Miyazaki Sciji , Uozumi Masanobu , Okamoto Naoko , Yamashita Shinji , Murakami Kazuo , Nakamura Yukio
Published: July 2002

Renin releasing stimulator, pro‐; Cortical slice; Submandibular gland; ELISA, prorenin‐prosegment; (Rat kidney)
Multiplicity of the DNA‐binding protein HBP‐1 specific to the conserved hexameric sequence ACGTCA in various plant gene promoters
Authors: Mikami Koji , Takase Hisabumi , Tabata Tetsuya , Iwabuchi Masaki
Published: July 2002

Multiplicity; DNA‐binding protein; Hexameric sequence; DNA‐protein interaction
Simple and rapid purification of F1ATPase from mitochondria
Authors: Shinohara Yasuo , Nakagawa Mariko , Terada Hiroshi
Published: July 2002

ATPase, F1; Purification, rapid; Dynosphere PD‐201‐SCX; Mitochondria; Oxidative phosphorylation
The mRNA transcripts from a mutant β‐globin gene derived from splicing at preferential cryptic sites
Authors: Lossi Anne-Marie , Bergé-Lefranc Jean-Louis
Published: July 2002

Thalassemia, β‐; mRNA splicing
Identification and purification of substrate‐binding subunit of higher plant H+‐translocating inorganic pyrophosphatase
Authors: Britten Christopher J. , Turner Janice C. , Rea Philip A.
Published: July 2002

Purification; Substrate‐binding subunit; Tonoplast; Vacuolar membrane; Pyrophosphatase, H+‐translocating inorganic
Expression of human granulocyte‐macrophage colony‐stimulating factor gene in insect cells by a baculovirus vector
Authors: Chiou Chuang-Jiun , Wu Ming-chi
Published: July 2002

A plasmid pAc373GM‐CSF was constructed and co‐transfected into Spodoptera frugiperda (Sf9) cells with wild‐type Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. The recombinant virus vAc373GM‐CSF was identified and purified by several rounds of plaque hybridization. By assaying the culture medium, we demonstrated recombinant virus infected Sf9 cells expressing hGM‐CSF. Recombinant hGM‐CSFs with apparent molecular masses of 14.5, 15.5 and 16.5 kDa were detected by the Western blot method. All 3 forms have biological activity of hGM‐CSF. Following N‐glycanase treatment, a single band of 14.5–15.5 kDa appeared in SDS‐PAGE. Western blot analysis of expression in Sf9 cells treated with tunicamycin revealed only the presence of the 14.5 kDa species. Thus, the signal sequence of recombinant hGM‐CSF could be recognized and cleaved by infected insect cell and the resultant molecule secreted into the media.