bluuebunny/crossref_metadata_2025 huggingface dataset:
Photolabelling of the prostaglandin E2 receptor in cardiac sarcolemmal vesicles
Authors: Michalak Marek , Wandler Elayne L. , Strynadka Ken , Lopaschuk Gary L. , Njue Wilson M. , Liu Hsing-Jang , Olley Peter M.
Published: July 2002

A [3H]azidophenacyl ester of PGE2 ([3H]azido‐PGE2) was synthesized and used to photoaffinity label the protein component of the high affinity PGE2 binding site in cardiac sarcolemma membrane. Photolysis of the isolated cardiac sarcolemmal vesicles in the presence of [3H]azido‐PGE2 resulted in the covalent labelling of a protein component that migrated on sodium dodecyl sulfate‐polyacrylamide gels with an apparent molecular weight of 100 000. Incorporation of the [3H]azido‐PGE2 did not occur in the absence of photolysis. The photolabelling of the 100‐kDa protein by [3H]azido‐PGE2 was inhibited by excess unlabelled PGE2 and arido‐PGE2. Specific binding of [3H]azido‐PGE2 was displaced by excess unlabelled PGE2 or azido‐PGE2, but not PGF2α, 6‐keto‐PGF1α or PGD2. These results indicate that the 100‐kDa photoaffinity labelled [3H]azido‐PGE2 binding protein contains the binding site for PGE2 in isolated cardiac sarcolemma membranes.
The effect of adenosine on the fluorescence responses of chlorotetracycline‐loaded human polymorphonuclear leukocytes
Authors: Tsuruta Satoru , Ito Setsuko , Mikawa Haruki
Published: July 2002

Chlorotetracycline has been used in human polymorphonuclear leukocytes as a probe to investigate the state of membrane‐bound calcium. We examined the effect of adenosine on the fluorescence responses of CTC‐loaded PMNs stimulated with the synthetic chemotactic peptide, formylmethionyl‐leucyl‐phenylalanine. Adenosine inhibited the decrease in CTC fluorescence in a dose‐dependent fashion and its effect was reversed by theophylline, an adenosine receptor antagonist. Removal of extracellular adenosine by incubating PMNs with adenosine deaminase abolished the effect of adenosine. These data suggest that adenosine inhibits the release of membrane‐bound calcium in PMNs that normally occurs in response to chemotactic stimuli, acting via PMN surface adenosine receptors.
Depression of prothymosin‐α production in murine thymus correlates with staphylococcal enterotoxin‐B‐induced immunosuppression
Authors: Low Teresa L.K. , Pan Tair-Long , Lin Yee-Shin
Published: July 2002

Prothymosin‐α (ProTα) and thymosin‐β4 (Tβ4) were isolated from murine thymus and characterized by microsequence analysis. Murine Tβ4 has an identical sequence to bovine Tβ4, whereas murine ProTα is highly homologous to rat ProTα. Murine ProTα differs from rat ProTα at two positions, Glu100 and Asp108 of the rat sequence are substituted by aspartic and glutamic acid, respectively, in murine ProTα. The amount of ProTα in murine thymus was found to be reduced after in vivo treatment with staphylococcal enterotoxin‐B (SEB), a superantigen which stimulates T cells bearing specific Vβ receptors. Results from the anti‐SRBC (sheep erythrocyte) plaque‐forming cell assay showed that the antibody response of the spleen cells from these animals was also suppressed. On the other hand, the amount of Tβ4 was not changed significantly. Our studies suggest that the suppression of SEB on antibody response correlates with the depression of ProTα production in the thymus.
A slowly inactivating calcium current works as a calcium sensor in calcitonin‐secreting cells
Authors: Scherubl H. , Schultz G. , Hescheler J.
Published: July 2002

Calcitonin (CT)‐secreting cells (C‐cells) are remarkably sensitive to changes in the extracellular Ca2+ concentration. In order to detect the mechanism by which C‐cells monitor Ca2+, we compared a C‐cell line responding to Ca2+ (rMTC cells) with another one known to have a defect in this Ca2+ signal transduction (TT cells). Rises of the Ca2+ concentration caused rMTC cells to depolarize and/or elicited spontaneous action potentials. Under voltage‐clamp conditions, rMTC cells showed a slowly decaying Ca2+ inward current which was sensitive to dihydropyridines but not to Ni2+ at a low concentration. In contrast, the ‘defective’ TT cells neither depolarized nor fired action potentials with high Ca2+; they only exhibited an Ni2+‐sensitive, transient Ca2+ current. The data strongly suggest that the slowly inactivating Ca2+ current is a prerequisite for Ca2+‐sensitivity of C‐cells and that fast inactivating channels are not sufficient to act as sensors of the extracellular Ca2+ concentration.
Transforming growth factor β and a mesoderm inducing factor from human blood platelets are different proteins
Authors: Dau Horst , Wedlich Doris , Born Jochen , Loppnow-Blinde Beate , Tiedemann Heinz , Tiedemann Hildegard
Published: July 2002

A mesoderm inducing factor from human blood platelets has been separated from transforming growth factor β and partially purified. The factor has similar chemical properties as mesoderm inducing factors from other sources.
GTPγS inhibits early c‐myc protein accumulation but not DNA synthesis in Swiss 3T3 fibroblasts
Authors: Pennington Stephen R. , Moore John P. , Evan Gérard I. , Hesketh T.Robin , Metcalfe James C.
Published: July 2002

Quiescent Swiss 3T3 fibroblasts stimulated with epidermal growth factor and insulin showed large transient increases in c‐myc mRNA and c‐myc protein accumulation which were maximal at about 2 h after addition of the co‐mitogens. When the cells were loaded with 0.1 mM of guanosine 5'‐O‐(3‐thiotriphosphate) (GTPγS) by transient permeabilisation immediately before mitogenic stimulation, the increase in c‐myc mRNA was similar to that observed in unloaded cells but the corresponding c‐myc protein peak was reduced by at least 95%. The GTPγS completely blocked incorporation of [35S]methionine into cell proteins for 3–4 h after addition of the mitogens, but not thereafter, and caused a delay in the subsequent onset of DNA synthesis by the same period. The data show that less than 5% of the early increase in c‐myc protein normally observed after mitogenic stimulation is required for its obligatory role in the progression of cells to S phase implied by other evidence.
cDNA encoding a 59 kDa homolog of ribosomal protein S6 kinase from rabbit liver
Authors: Harmann Beate , Kilimann Manfred W.
Published: July 2002

We have isolated cDNA molecules encoding a protein with the characteristic sequence elements that are conserved between the catalytic domains of protein kinases. This protein is apparently a serine/threonine kinase and is most closely related to the amino‐terminal half of the ribosomal protein S6 kinase II first characterized in Xenopus eggs (42% overall identity and 56% identity in the predicted catalytic domain). However, it clearly differs from S6 kinase II in that it has only one, rather than two predicted catalytic domains and a deduced molecular mass of 59109 Da. We propose that is may be more related to, or identical with, the mitogen‐inducible S6 kinase of molecular mass 65–70 kDa described in mammalian liver, mouse 3T3 cells and chicken embryos. Remarkable structural features of the cDNA‐encoded polypeptide are a section rich in proline, serine and threonine residues that resemble the multiphosphorylation domains of glycogen synthase and phosphorylase kinase a subunit, and a characteristic tyrosine residue in the putative nucleotide‐binding glycine cluster which, by analogy to cdc2 kinase, is a potential tyrosine phosphorylation site.
Modulation of calcium mobilization by guanosine 5'‐O‐(2‐thiodiphosphate) in Xenopus oocytes
Authors: Sealfbn Stuart C. , Mundamattom Shirly , Gillo Boaz
Published: July 2002

We investigated the effect of intracellular loading of the non‐hydrolyzable guanosine 5'‐diphosphate analogue GDPßS on calcium mobilization by IP3 and on calcium influx in Xenopus oocytes. Assayed by two electrode voltage‐clamp recording, GDPßS‐loaded oocytes demonstrated a marked augmentation of the fast component of the response to IP3 injection, an attenuation of the slow component and an increase in membrane calcium permeability. These effects on calcium mobilization suggest that gdpβs may facilitate calcium translocation both across the plasma membrane and between different intracellular calcium pools.
Anti‐idiotypic antibody identifies the structural similarity between the phosphatidylcholine‐specific monoclonal antibody and phosphatidylcholine‐specific lipid transfer protein
Authors: Nam Kyung Soo , Umeda Masato , Igarashi Kouji , Inoue Keizo
Published: July 2002

The polyclonal anti‐idiotypic antibody (Anti‐Id) has been raised against a monoclonal antibody (mAb) that specifically binds to phosphatidylcholine (PC). The anti‐Id bound strongly to PC‐specific mAbs, but not to the other mAbs that bind to phosphatidylserine, indicating that the anti‐Id recognizes the cross‐reactive idiotopes expressed on the PC‐specific mAbs. The anti‐Id also showed an extensive cross‐reaction with the PC‐specific lipid transfer protein isolated from bovine liver and inhibited the lipid transfer activity of the protein. These results strongly suggest that the anti‐Id recognizes a common structure shared between PC‐specific mAbs and the PC‐specific lipid transfer protein.
Characterization of cucumber mosaic virus RNA‐dependent RNA polymerase
Authors: Quadt R. , Jaspars E.M.J.
Published: July 2002

Cucumber mosaic virus (CMV) RNA‐dependent RNA polymerase (RdRp) was purified form CMV‐infected tobacco. The purified enzyme is completely dependent an exogenous template. The enzyme ultilizes a variety of viral RNAs and CMV satellite RNA as template for minus‐strand synthesis. Cellular RNAs are not used as templates. Ribosomal RNA inhibits the viral RNA synthesis by the CMV RdRp.
Differential effect of extracellular calcium on the Na+ ‐K+ pump activity in intact polymorphonuclear leucocytes and erythrocytes
Authors: Petersen Reno H. , Knudsen Torben , Johansen Torben
Published: July 2002

The effect of extracellular calcium on the Na+ ‐K+ pump activity in human polymorphonuclear leucocytes and erythrocytes was studied and compared with the activity in mixed peritoneal leucocytes from rats. While there was maximal decrease in the pump activity (25–30%) of leucocytes from both rat and human by calcium 0.6 mM, a concentration of 0.1 mM caused a substantial decrease indicating a high sensitivity for extracellular calcium. In contrast, calcium had no effect on the pump activity in crythrocytes. The effect of calcium on the pump activity in leucocytes may be due to regulation of the influx of sodium across the plasma membrane, since in human leucocytes calcium had no effect on the pump activity if the cells were loaded with sodium.
NADH:ubiquinone oxidoreductase from bovine heart mitochondria Complementary DNA sequence of the import precursor of the 10 kDa subunit of the flavoprotein fragment
Authors: Skehel J.Mark , Pilkington Stephanie J. , Runswick Michael J. , Fearnley Ian M. , Walker John E.
Published: July 2002

The amino acid sequence of the 10 kDa subunit of the flavoprotein (FP) fragment of complex I from bovine heart mitochondria has been determined by protein sequence analysis, thereby completing the sequence of the FP fragment. The calculated molecular weight of the 10 kDa subunit agrees exactly with the value of 8438 determined by electrospray mass spectrometry, and further confirmation of the sequence has been obtained by sequencing cDNA amplified from total bovine heart cDNA by the polymerase chain reaction, using mixed oligonucleotides based upon the protein sequence as primers and hybridization probes. The sequence of the 10 kDa subunit is not related to that of any known protein. Being devoid of cysteine residues, it has none of the characteristic features of known iron‐sulfur proteins and it is improbable that it is involved in liganding Fe‐S centers in the FP fragment.
Simple approach for oligonucleotide‐directed mutagenesis of any double‐stranded circular DNA
Authors: Drutsa V.L. , Kaberdin V.R.
Published: July 2002

A new site‐directed method for introducing mutations into any region of plasmid vector close to the unique restriction site is described. It is based on the use of 5′‐phosphorylated mutagenic and nonphosphorylated auxiliary oligonucleotides and a specific combination of enzymatic procedures including ‘nick‐translation’ as a key step. The method efficiency was demonstrated by constructing the deletion‐insertion mutation wich creates the consensus Pribnow box in a promoter‐testing plasmid. The yield of the target mutation was up to 85–95%.
Metaphase chromosomes from mammalian cells stimulate fusion of artificial phospholipid vesicles
Authors: Balakireva L.A. , Belyaev N.D. , Budker V.G. , Kiseleva E.V.
Published: July 2002

Isolated mitotic chromosomes are able to form complexes with phosphatidylcholine liposomes in the presence and absence of Ca2+ ions, in the latter case in the presence of polyamines. Interactions with chromosomes stimulates liposome fusion. The fusion is promoted by condensed and EDTA‐decondensed chromosomes.
Preliminary X‐ray crystallographic analysis of tryptophanase from Escherichia coli
Authors: Kawata Yasushi , Tani Shunsuke , Sato Mamoru , Katsube Yukiteru , Tokushige Masanobu
Published: July 2002

Tryptophanase (L‐tryptophan indole‐lyase) from Escherichia coli has been crystallized from ammonium sulfate solution using a vapor diffusion method. The crystals are tetragonal and belong to space group P41212 or its enantiomorph. The cell dimensions of the crystals are a = b = 113.4 Å, and c = 232.2 Å, with two subunits per asymmetric unit. The crystals diffract to at least 3 Å resolution, and are suitable for X‐ray structural analysis.
Evidence that reverse cholesterol transport is stimulated by lipolysis of triglyceride‐rich lipoproteins
Authors: Miller N.E. , Nanjee M.N.
Published: July 2002

The hypothesis that reverse cholesterol transport by high density lipoprotein (HDL) is augmented by lipolysis of triglyceride‐rich lipoproteins received support from experiments in rabbits whose tissue cholesterol had been pre‐labeled with [3H]cholesterol several weeks earlier. When lipolysis was stimulated by intravenous heparin (which releases lipoprotein lipase from vascular endothelium), reciprocal changes in plasma triglyceride and HDL cholesterol concentrations were accompanied by a rise in the specific radioactivity of HDL cholesterol, indicative of increased transfer of cholesterol into HDL from slowly exchanging cholesterol pools in extra‐hepatic tissues.
Purification and partial characterization of ubiquitin‐activating enzyme from Saccharomyces cerevisiae
Authors: Hoefer Markus , Cook James C.
Published: July 2002

Ubiquitin‐activating enzyme was purified from the yeast Saccharomyces cerevisiae by covalent affinity chromatography on ubiquitin‐Sepharose followed by HPLC anion‐exchange chromatography. Enzyme activity was monitored by the ubiquitin‐dependent ATP: 32PP1 exchange assay. The purified enzyme has a specific activity of 1.5μmol 32PP1 incorporated into ATP·min−1·mg−1 at 37°C and pH 7.0 under standard conditions for substrate concentrations as described by Ciechanover et al. (1982) J. Biol. Chem. 257, 2537–2542. The catalytic activity showed a maximum at pH 7.0. Its molecular weight both in non‐denaturing and in SDS‐gel electrophoresis was estimated to be 115 kDa, suggesting a monomeric form. The isoelectric point determined by gel electrofocusing was approximately 4.7. Two protein bands differing slightly in electrophoretic mobility could be distinguished when SDS gels were loaded with very small amounts of purified E1 and immunoblotted, the one with higher molecular weight being clearly predominant. The same two bands were also found in anti‐E1 immunoblots of crude yeast lysates prepared under broad protease inhibition.
Immunological characterization of peptidyl‐glycine‐α‐amidating monooxygenases
Authors: Sharoyan S.G. , Nalbandyan R.M.
Published: July 2002

Antibodies to the soluble form of the copper‐containing enzyme, peptidyl‐glycine‐α‐amidating monooxygenase isolated from secretory granules of bovine pituitary anterior lobes were found to belong to immunoglobulin G1. The antibodies were used to study the subcellular distribution of the enzyme in this tissue, and positive tests were found only for granular and cytosol fractions. The antibodies do not crossreact with other copper‐containing systems of secretory granules, such as neurocuprein and dopamine‐ß‐monooxygenase. It was shown that the antibodies give the crossreaction with the enzyme isolated from secretory granules of bovine pituitary anterior lobes, cardiac atria, pancreas and adrenal medulla, indicating the antigenic identity of the enzyme from secretory granules of different glands.
Immunochemical identification of branched‐chain 2‐oxo acid dehydrogenase kinase
Authors: Shimomura Yoshiharu , Nanaumi Noriko , Suzuki Masashige , Harris Robert A.
Published: July 2002

Branched‐chain 2‐oxo acid dehydrogenase kinase was characterized using anti‐kinase polygonal antibodies. The antibodies were purified from rabbit antiserum by an epitope selection method. The antibodies bound only to a 44 kDa polypeptide in the dehydrogenase—kinase complex and inhibited the kinase activity, substantiating that the 44 kDa polypeptide is the catalytic subunit of the kinase. The purified liver dehydrogenase—kinase complex, but not either the purified heart complex or the partially purified liver complex, contained 2 additional polypeptides of lower molecular weight which also reacted with the anti‐kinase antibodies, suggesting that the liver kinase is subject to proteolytic degradation during purification.
Proton dissociation from nigericin at the membrane—water interface, the rate‐limiting step of K+/H+ exchange on the bilayer lipid membrane
Authors: Kovbasnjuk Olga N. , Antonenko Yuri N. , Yaguzhinsky Lev S.
Published: July 2002

The rate of K+/H+ exchange through bilayer lipid membranes (BLM) induced by nigericin was measured by the method of pH gradient offset according to Antonenko, Yu.N. and Yaguzhinsky L.S. [(1990) Biochim. Biophys. Acta 1026, 236–240]. It was shown that under the conditions of high potassium ion concentration the rate of nigericin‐mediated K+/H+ exchange increased with an increase in the concentrations of such buffer compounds as citric acid and MES. The concentration dependence was different for citrate and MES. The buffer concentration effect was absent at low potassium ion concentrations. Citrate increased the rate of K+/H+ exchange being added to the side of BLM where the K+ concentration was higher and had no effect at the opposite side. At High KCl and citrate concentrations, the rate of K+/H+ exchange was about 6 times lower in D2O when compared to H2O solutions. It is concluded that under certain experimental conditions the overall rate of the K+/H+ exchange induced by nigericin is determined by the rate of proton dissociation from nigericin at the membrane—water interface.
Reconstitution of the solubilized cardiac sarcoplasmic reticulum potassium channel Identification of a putative Mr ∼80 kDa polypeptide constituent
Authors: Liu Qi-Yi , Lai F.Anthony , Shen Win K. , Meissner Gerhard , Strauss Harold C.
Published: July 2002

Recent evidence has indicated that potassium ion movement through sarcoplasmic reticulum (SR) K+ channels is an important countercurrent for Ca2+ release from SR. We used Chaps‐solubilized SR vesicles and sucrose density gradient centrifugation to identify components of the canine cardiac SR K+ channel. To overcome the difficulty of the absence of a high‐affinity specific ligand, we have successfully applied the planar lipid bilayer reconstitution technique to identify and functionally assay for the solubilized SR K+ channel. We found that Chaps solubilization of the channel did not change the protein's functional properties. The cardiac SR K+ channel sediments as a 15–20S protein complex. A polypeptide of M
r ∼80 kDa was found to specifically comigrate with the 15–20S gradient fractions and might be a major constituent of the cardiac SR K+ channel.
Phytochrome control and anoxia effect on the activity and expression of soybean seedling lipoxygenases 1 and 2
Authors: Maccarrone Mauro , Veldink Gerrit A. , Vliegenthart Johannes F.G.
Published: July 2002

The effects of different light irradiations and anoxia treatment on the activity and expression of the lipoxygenases 1 (LOX‐1) and 2 (LOX‐2) of soybean seedlings have been investigated. A phytochrome‐mediated decrease of the specific activities of both isoenzymes was found which was due to the regulation of the gene expression at the transcriptional level. The anoxia treatment of the seedlings led to a decrease of both lipoxygenase specific activities as well, but this effect was attributable to the reduction of the amount of the two isoenzymes, without changes in the steady‐state levels of specific mRNAs. Therefore, light and anoxia are both regulators of soybean lipoxygenases, but their mechanism of action at the molecular level is different.
Relative‐residue surface‐accessibility patterns reveal myoglobin and catalase similarity
Authors: Cockcroft V.B. , Osguthorpe D.J.
Published: July 2002

A novel sliding‐window search method using relative‐residue surface‐accessibility patterns identified extensive, but unsuspected, structural similarity over a 3‐helix region in the C‐terminus of the evolutionarily unrelated proteins sperm‐whale myoglobin and beef liver catalase. This clear example of structural similarity between non‐homologous proteins highlights the importance of relative‐residue surface‐accessibility patterns in understanding the local folded structure in proteins.
Identification of some of the brain Gn27 as the ral gene product Comparison between the brain and platelet Gn‐proteins
Authors: Bhullar Rajinder P.
Published: July 2002

Two major Gn‐proteins, Gn27 and Gn26, were detected in the 100,000 × gav particulate fraction of rabbit and bovine brain. The Gn26 protein was also present in significant amounts (∼50% of total) in the brain supernatant fraction. An antiserum raised against recombinant simian ralA recognized a 27‐kDa brain protein with the same apparent molecular mass as the Gn27 protein. In further analysis by two‐dimensional polyacrylamide gel electrophoresis, the brain particulate Gn‐proteins were resolved into 6 major forms, four of 27 kDa (Gn27a–d) and two of 26 kDa (Gn26a and Gn26b). Minor GTP‐binding components were also observed at 25 kDa and 24 kDa. The ratA antibody reacted strongly with the brain Gn27b form and weakly with the Gn27a and Gn27c but not with Gn27d or any of the other Gn‐proteins. In addition, comparison of human platelet and bovine brain particulate Gn‐proteins by two‐dimensional polyacrylamide gel electrophoresis demonstrated a tissue/cell‐type specific expression of the various forms of Gn‐proteins.
Superproduction of heavy minicircular mitochondrial DNA in aging wheat coleoptiles
Authors: Kirnos M.D. , Alexandrushkina N.I. , Zagorskaya G.Ya. , Kireev I.I. , Vanyushin B.F.
Published: July 2002

On incubation of 7‐day‐old wheat seedlings in the presence of [3H]thymidine, the radioactivity incorporated into coleoptile DNA is found to be localized mainly (>95%) in the fraction of heavy mitochondrial DNA (H‐mt DNA: ϱ = 1.716 gm/cm3). Upon long (48–72 h) incubation of cut‐off seedlings in water, the amount of this DNA shows a dramatic increase corresponds to about 10% of the total coleoptile DNA. H‐mtDNA is represented by open circular molecules with a contour length varying from 0.12 to 0.6μm. The functional role of this DNA is still unknown.
Developmental changes in erythropoietin receptor expression of fetal mouse liver
Authors: Masuda Seiji , Hisada Yutaka , Sasaki Ryuzo
Published: July 2002

Erythropoietin (EPO) stimulates proliferation and differentiation or late erythroid precursor cells (CFU‐E) and thereby determines the rate of erythropoiesis, Liver is the major erythropoietic site in a fetus. We dealt with developmental changes in CFU‐E and EPO receptor (EPO‐R) of fetal mouse liver. The affinity of the EPO‐R to EPO was unchanged during fetal development. The popupation size of CFU‐E, the number of EPO‐R per liver cell, and EPO‐R mRNA decreased as gestation proceeded, in a pattern indicating that the expression of EPO‐R on erythroid precursor cells in fetal mouse liver is governed mostly by the process of mRNA production.
Localisation of the [32P]IP3 binding site on human platelet intracellular membranes isolated by high‐voltage free‐flow electrophoresis
Authors: Authi Kalwant S.
Published: July 2002

This Study reports the localisation of the [32P]IP3 binding site on highly purified membrane fractions prepared using high‐voltage free‐flow electrophoresis. Binding studies on mixed membranes, carried out at 4°C, revealed a binding site with a K
d = 86 nM and βmax = 5.3 pmol/mg protein. The binding was potently inhibited by heparin. High‐voltage free‐flow electrophoresis was used to further purify surface and intracellular membranes. The intracellular membranes showed a 5‐fold enrichment of binding sites with respect to the parent mixed membranes with the same K
d(80 nM), but the surface membranes showed an absence of binding activity. The results indicate the localisation of the IP3 receptor on highly purified intracellular membranes.
Prostaglandin F2α decreases the affinity of epidermal growth factor receptors in Swiss mouse 3T3 cells via protein kinase C activation
Authors: de Asua Luis Jimenez , Goin Mercedes
Published: July 2002

Prostaglandin F2α (PGF2α) selectively decreases the binding of 125I‐labelled epidermal growth factor ([125I]EGF) to intact Swiss 3T3 cells. Scatchard analysis reveals that PGF2α decreases the number of high‐affinity EGF binding sites without changing the total number of receptors. Prostaglandins E1 (PGE1), E2 (PGE2 or F2β (PGF2β) do not alter the EGF binding to these cells and do not enhance the PGF2α effect. R‐59022 and R‐59949, two diacylglycerol kinase inhibitors, enhance the inhibitory effect of PGF2α, whereas down‐modulation of protein kinase C (PKC) abolishes the effect. These results indicate that PGF2α, decreases EGF binding in Swiss 3T3 cells via PKC activation.
Accumulation of a translation intermediate of DI protein by light—dark transition in isolated spinach chloroplasts
Authors: Inagaki Noritoshi , Satoh Kimiyuki
Published: July 2002

In an in vitro translation experiment using spinach chloroplasts a novel protein band of about 17.5 kDa appeared by light to dark transition. The protein never accumulated in detectable amounts either in continuous illumination or in continuous darkness. The 17.5 kDa protein accumulated upon light—dark transition, on the other hand, disappeared by the subsequent illumination. Accumulation of the protein in light, however, was observed when stromal level of ATP in chloroplasts was lowered after preillumination by the addition of various chemical compounds which, irrespective of the mode of action, eventually decrease the ATP level, e.g. atrazine, carbonyl‐cyanide‐?‐chlorophenyl hydrazone and glycerate. The dark‐accumulated protein was concluded to be a translation intermediate of DI protein based on the facts that this component precipitates with specific antibodies and is resistant to lyxylendopeptidase treatment. The suppression by chloramphenicol of both appearance upon light‐dark transition and disappearance by the subsequent illumination of the protein also supported this conclusion. The phenomenon was discussed in terms of pausing in the translation of psbA mRNA
Identification of a component separated on Mono Q purification of Escherichia coli RNA polymerase as an NTPase
Authors: Butzow James J. , Stankis Rosemary G.
Published: July 2002

Standard preparations of Escherichia coli RNA polymerase (RNAP) contain NTPase activity. High‐performance anion exchange chromatography on Mono Q has recently been used by Hager et al. [1990, Biochemistry 29, 7890–7894] to fractionate RNAP into holoenzyme (α,ββ′σ) and core (α,ββ′) forms, plus other protein components. We found that one of these components, of protomer size slightly larger than the σ70 subunit, has NTPase activity; it is efficiently separated on Mono Q, leaving transcriptionally active holoenzyme and core apparently free of NTPase activity. Because of the similarity in size with σ70, the NTPase component may escape detection by routine gel electrophoresis.
Reduction of carbon monoxide to formaldehyde by the terminal oxidase of the marine bacterium Pseudomonas nautica strain 617
Authors: Arnaud Sylvain , Malatesta Francesco , Denis Michel
Published: July 2002

When exposed to CO, the aerobic respiratory system of the marine bacterium Pseudomonas nautica strain 617, previously reduced with dithionite, undergoes reoxidation. When dealing with the purified oxidase (dithionite reduced) exposure of the enzyme to CO induces its reoxidation (collapse of its α band). Under our experimental conditions, this form of the oxidase could not be reduced again by dithionite. Addition of formaldehyde to the native oxidized enzyme resulted in full inhibition of the oxidase reduction by dithionite, presumably due to complex formation. We hypothesized a reduction of CO into formaldehyde and a locking of the active site by the reaction product. By using flash photolysis, it was possible to turn over the enzyme, accumulate the reaction product and identify it as formaldehyde. When using the membrane‐bound enzyme, formaldehyde accumulated without the help of flash photolysis. This unusual reduction of CO to formaldehyde could be related to the previously reported uncommon features of the P. nautica oxidase, in particular O2 reduction into H2O2 as end product [(1989) FEBS Lett. 247, 475–479].
ADP‐ribosylation by Clostridium botulinum C3 exoenzyme increases steady‐state GTPase activities of recombinant rhoA and rhoB proteins
Authors: Mohr Christiane , Koch Gertrud , Just Ingo , Aktories Klaus
Published: July 2002

ADP‐ribosylation of recombinant rhoA and rhoB proteins by Clostridium botulinum C3 exoenzyme increased steady‐state GTP hydrolysis by 50 to 80%. ADP‐ribosylation and increase in GTP hydrolysis occurred at similar concentrations of C3, depended on the presence of NAD and were prevented by anti‐C3 antibody or heat inactivation of C3. In contrast, GTP hydrolysis by Ile‐41 rhoA or Ha‐ras, which are no substrates for the transferase, were not affected by C3. ADP‐ribosylation facilitated the [3H]GDP release and subsequently, the binding of [3H]GTP to rhoA. The data indicate that the increase in the steady‐state GTPase activity by ADP‐ribosylation is caused by increasing the rate of GDP release which is suggested to be the rate limiting step of the GTPase cycle of the small GTP‐binding proteins.
Functional and immunological relationships between metyrapone reductase from mouse liver microsomes and 3α‐hydroxysteroid dehydrogenase from Pseudomonas testosteroni
Authors: Maser Edmund , Oppermann Udo C.T. , Bannenberg Gudula , Netter Karl J.
Published: July 2002

3α‐Hydroxysteroid dehydrogenase (3α‐HSD) from Pseudomonas testosteroni was shown to reduce the xenobiotic carbonyl compound metyrapone (MPON). Reversely, MPON reductase purified from mouse liver microsomes and previously characterized as aldehyde reductase, was competitively inhibited by 3α‐HSD steroid substrates. For MPON reduction both enzymes can use either NADH or NADPH as co‐substrate. Immunoblot analysis after native and SDS gel electrophoresis of 3α‐HSD gave a specific crossreaction with the antibodies against the microsomal mouse liver MPON reductase pointing to structural homologies between these enzymes. In conclusion, there seem to exist structural as well as functional relationships between a mammalian liver aldehyde reductase and prokaryotic 3α‐HSD. Moreover, based on the molecular weights and the co‐substrate specificities microsomal mouse liver MPON reductase and Pseudomonas 3α‐HSD seem to be members of the short‐chain alcohol dehydrogenase family.
Specific inhibition of binding of antistasin and [A103,106,108] antistasin 93–119 to sulfatide (Gal(3‐SO4)β1‐1Cer) by glycosaminoglycans
Authors: Brankamp Robert G. , Manley George D. , Owen Thomas J. , Krstenansky John L. , Cardin Alan D.
Published: July 2002

Leech‐derived antistasin is a potent anticoagulant and antimetastatic protein that binds sulfatide (Gal(3‐SO4)β1‐1Cer)and sulfated polysaccharides. In this study, the synthetic fragment [A103,106,108] antistasin 93–119, which corresponds to the carboxyl terminus, showed specific and saturable binding to sulfatide. Binding was competitively blocked by glycosaminoglycans (GAGs) in the order: dextran sulfate 5000 ≅ dextran sulfate 500 0OO > heparin > dermatan sulfate ⪢ chondroitin sulfates A and C. This rank order of inhibitory potency was identical to that observed with whole antistasin. We suggest that residues 93–119 of antistasin represent a critical domain for binding GAGs and sulfated glycolipids.
Interaction of ACTH, β‐endorphin and α‐melanocyte stimulating hormone in relation to the corticosteroid production of isolated rat adrenocortical zona fasciculata and zona glomerulosa cells
Authors: Szalay Katalin Sz. , Folly Gábor
Published: July 2002

The combined effects of ACTH, β‐endorphin (β‐EP) and α‐MSH were studied on the corticosteroidogenesis or isolated rat adrenocortical zona fasciculata and zona glomerulosa cells. β‐EP potentiated the effects of ACTH and α‐MSH on the zona fasciculata corticosterone production but inhibited those on the zona glomerulosa aldosterone production. β‐EP did not affect the combined action of 4×10−11 M ACTH and 5×10−9 M α‐MSH on the zona fasciculata or the zona glomerulosa cells, but it inhibited the stimulatory action of the combination of 1.6×10−10 M ACTH and 10−9 M α‐MSH on the zona glomerulosa aldosterone production. An interaction of ACTH, β‐EP and α‐MSH in relation to the zona fasciculata and zona glomerulosa corticosteroid production was found.
Fluorescence study of the nucleic acid binding site of vimentin
Authors: Kooijman Martin , Bloemendal Michael , Traub Peter , van Grondelle Rienk
Published: July 2002

A selective tyrosine fluorescence quenching is found on interaction of vimentin with poly(dT) and poly(rA). However, addition of poly(dA) does not result in tyrosine quenching. The number of nucleotides covered by vimentin upon binding(n) of poly(dT) (50 ± 4) appeared to be approximately the same as for poly(rA) (44 ± 4), while the apparent binding constant (K
app) of the latter is slightly larger (5.0 ± 2.0 × 107 M−1·cm−1 vs. 2.5 ± 0.5 × 107 M−1·cm−1). The finding that there exists a specific strong interaction between vimentin and nucleic acids could help in the search for cellular functions of intermediate filament proteins.
Upstream activation element of the PH03 gene encoding for thiamine‐repressible acid phosphatase in Saccharomyces cerevisiae
Authors: Nosaka K. , Yamanishi K. , Nishimura H. , Iwashima A.
Published: July 2002

The PH03 gene of Saccharomyces cerevisiae encodes thiamine‐repressible acid phosphatase and requires the positively acting regulatory protein TH12 for its expression. Deletion analysis of the 5′‐flanking region of PH03 gene revealed that an activating region located at nucleotide position −234 to −215 relative to the translation initiation codon is required for the expression and sensitivity to thiamine. A chemically synthesized DNA fragment covering −234 to −215 showed a significant level of expression when inserted in front of the PH03 promoter lacking the activating region. Electrophoretic mobility shift assay demonstrated the presence of proteins that bound to the above DNA fragment in the nuclear extract from cells grown in thiamine‐free medium. These findings suggested that this region between −234 and −215 acts as an upstream activation element of the PH03 gene that can interact with regulatory proteins.
Nucleotide composition of genes and hydrophobicity of the encoded proteins
Authors: Mázek Jan , Kypr Jaroslav
Published: July 2002

We find that true proteins are generally more hydrophobic than the corresponding hypothetical proteins encoded by the randomized gene nucleotide sequences. Furthermore, the protein hydrophobicity but not its gene nucleotide composition is conserved within evolutionary families of functionally related proteins. These two findings indicate that there is a general drift to modify gene nucleotide composition in the course of evolution. An inspection of codon usage in genes shows that the drift mainly increases the content of adenine at the expense of thymine.
α2‐Macroglobulin and other proteinase inhibitors do not interfere with the secretion of amyloid precursor protein in mouse neuroblastoma cells
Authors: De Strooper Bart , Van Leuven Fred , Van Den Berghe Herman
Published: July 2002

A series of proteinase inhibitors active against proteinases of all four major classes, including highly purified and well‐characterized α2‐macroglobulin, added to the cell culture medium of murine Neuro 2a neuroblastoma cells did not interfere with APP secretase activity. We therefore advance the hypothesis that APP secretase activity is localized in an intracellular compartment.
Identification of heme macrocycle type by near‐infrared magnetic circular dichroism spectroscopy at cryogenic temperatures
Authors: Peng Qinyun , Timkovich Russell , Loewen Peter C. , Peterson Jim
Published: July 2002

The electron paramagnetic resonance (EPR) and near‐infrared magnetic circular dichroism (MCD) spectra of the azide and cyanide adducts of nitrimyoglobin and hydroperoxidase II from Escherichia coli have been measured at cryogenic temperatures. For the first time, ligand‐to‐metal charge‐transfer transitions in the near‐infrared have been observed for an Fe(III)‐chlorin system. It is shown that near‐ultraviolet‐to‐visible region electronic spectra of ‘green’ hemes such as these are an unreliable indicator of macrocycle type. However, the combined application of EPR and near‐infrared MCD spectroscopics clearly distinguishes between the porphyrin‐containing nitrimyoglobin and the chlorin‐containing hydroperoxidase II.
Vanadate potentiates the glycogenic action of insulin‐like growth factors on isolated diaphragm
Authors: Vandorpe Greet , Bollen Mathieu , Van Herck Erik , Bouillon Roger , Stalmans Willy
Published: July 2002

Na3VO4 (6.5 μmol/100 g rat weight), co‐injected with a trace amount of [???C]glucose, increased within 15 min the incorporation of radiolabel in diaphragmal glycogen. After 2 h the vanadate‐induced increases were 12‐fold in the diaphragm and 7–8‐fold in heart and liver. In contrast, when added to isolated diaphragms for up to 1 h, vanadate (0.1–5 mM) had no effect on the synthesis of glycogen from 5 mM glucose. In search of a putative mediator of vanadate's action in vivo, insulin and the insulin‐like growth factors (IGFs) were considered. Their plasma concentration was not affected by vanadate treatment. In isolated diaphragms, 1 mM vanadate did not potentiate insulin‐induced glycogen synthesis, but it caused a several‐fold increase in glycogen synthesis in the presence of concentrations of IGF‐I which, alone, had no effect. A similar synergism occurred between vanadate and IGF‐II. We propose that the glycogenic action of vanadate in vivo, at least in some tissues, involves a potentiation of the action of IGF‐I.
m‐Iodobenzylguanidine increases the mitochondrial Ca2+ pool in isolated hepatocytes
Authors: Juedes Marlene J. , Kass George E.N. , Orrenius Sten
Published: July 2002

The incubation of isolated hepatocytes with the inhibitor of protein mono ADP‐ribosylation, m‐iodobenzylguanidine (MIBG), resulted in an increase in the size of the mitochondrial Ca2+ pool, without alteration of the non‐mitochondrial Ca2+ store(s). This increase was abolished when the cytosolic free Ca2+ concentration ([Ca2+]i) was buffered by prior loading of the cells with fluo 3. Elevating [Ca2+]i by releasing the endoplasmic reticular Ca2+ store with 2,5‐di‐(tert‐butyl)‐1,4‐hydroquinone resulted in a synergistic increase in the magnitude of the mitochondrial Ca2+ pool. A role for protein ADP‐ribosylation in the intracellular regulation of mitochondrial Ca2+ homeostasis is suggested.
Folylpolyglutamate analogs can inhibit casein kinase II from Xenopus laevis
Authors: Tellez Rowena , Allende Catherine C. , Allende Jorge E.
Published: July 2002

Polyglutamate analogs of folate and related compounds were tested as inhibitors of casein kinase II (CK II) obtained from Xenopus laevis. The inhibitory capacity of the pteroyl, 4‐amino‐10‐methyl pteroyl (the methotrexate aromatic moiety), and p‐aminobenzoil derivatives increased as the the number of γ‐glutamates attached went from 2 to 7. The nature of the aromatic head group was also important since hexa‐γ‐glutamatic acid had no inhibitory activity while the folylhexaglutamate derivatives were strong inhibitors with relative potency of methotrexate > pteroyl >
p‐aminobenzoic acid. The inhibition of CK II by methotrexate γ‐pentaglutamate was competitive with casein and showed an apparent K
i of 90 μM.
The protein phosphatase inhibitor, okadaic acid, potentiates the stimulatory effect of phorbol ester on phosphatidylcholine synthesis, but not on phospholipid hydrolysis, in fibroblasts
Authors: Kiss Zoltan
Published: July 2002

The potent protein phosphatase inhibitor, okadaic acid, was used to determine the possible role of protein phosphorylation reaction(s) in phorbol ester‐induced synthesis and hydrolysis of phosphatidylcholine (PtdCho) in NIH 3T3 fibroblasts. Okadaic acid (2μM) was found to enhance the stimulatory effects of lower concentrations (2.5–25 nM) of phorbol 12‐myristate 13‐acetate (PMA) on PtdCho synthesis, but not an PtdCho hydrolysis, after treatments for 30–60 min. These data support a view that in fibroblasts PMA simulates only PtdCho synthesis, and not PtdCho hydrolysis, by a protein phosphorylation‐dependent mechanism.
Changes in Na+,K+‐ATPase structure induced by cation binding Approach by Raman spectroscopy
Authors: Anzenbacher Pavel , Mojzes Peter , Baumruk Vladimir , Amler Evžen
Published: July 2002

Raman analysis of Na+, K+‐ATPase structural changes induced by cation binding reveals a slight decrease (< 10%) of the α‐helical content upon E1–E2 transition. Pronounced conformational changes of the enzyme are unlikely as the character of the environment of tyrosine residues remains unaltered. However, local changes can take place as evidenced by changes in tryptophan vibration at about 880 cm−1.
Interaction of α subunit of GTP‐binding protein Go with a 20‐kDa Triton‐insoluble membrane protein in bovine brain
Authors: Shiozaki Tateki , Negishi Manabu , Takahashi Satoru , Ichikawa Atsushi
Published: July 2002

Heterotrimeric Go bound to the membranes of bovine brain, but Goα remained bound to the membranes even after activation with GTPγS. Furthermore, Goα bound to a Triton X‐100‐insoluble fraction of the membranes in a saturable manner. However, the 37‐kDa Goα eliminated by trypsin at the amino‐terminus could not bind to the fraction. Using a blot overlay approach of the Triton‐insoluble fraction, only a 20‐kDa protein was identified that interacts with Goα. These results indicate that Goα binds to a 20‐kDa Triton‐insoluble protein in the bovine brain membranes.
Full activation without calmodulin of calmodulin‐dependent cyclic nucleotide phosphodiesterase by acidic glycosphingolipids: GM3, sialosylneolactotetraosylceramide and sulfatide
Authors: Higashi Hideyoshi , Yamagata Tatsuya
Published: July 2002

Among calmodulin‐non‐binding glycosphingolipids, GM3, sialosylneolactotetraosylceramide (LM1), and sulfatide potently activated calmodulin‐dependent cyclic nucleotide phosphodiesterase with or without Ca2+ showing ED50 1–5 μM. In contrast to calmodulin‐binding gangliosides, these glycosphingolipids activated the enzyme up to the maximum level achieved by Ca2+/calmodulin and did not inhibit the activity at higher concentrations. Competition studies with GD1b that bind both to calmodulin and the enzyme suggest that the calmodulin‐non‐binding glycosphingolipids activate the enzyme through interaction with the same site of the enzyme as GD1b interacts.
Identification of CpG island at the 5' end of murine leukemia inhibitory factor gene
Authors: Kašpar Petr , Dvořák Michal , Bartůněk Petr
Published: July 2002

We identified a CpG island at the 5' end of murine leukemia inhibitory factor gene (LIF). The CpG island is 0.6 kb long and covers most of the first exon and first intron. The region is non‐methylated, its G+C content is 65% and relative frequency of CpG dinucleotide is 0.7. The block of 150 nucleotides, which is 72% conserved between murine, human, ovine and porcine genes, is a part of the CpG island. Two DNA fragments from this CpG island interact with nuclear proteins from NIH 3T3 cells. One fragment partially covers the block of conserved nucleotides. Human, ovine and porcine LIF genes also contain G+C‐ and CpG‐rich sequences in the corresponding region.
Reduced glutamate decarboxylase activity in rat islet β cells which survived streptozotocin‐induced cytotoxicity
Authors: Ling Z. , Malaisse-Lagae F. , Malaisse W.J. , Pipeleers D.
Published: July 2002

Rat pancreatic β cells exhibit a 16‐fold higher glutamate decarboxylase (GAD) activity than islet non‐β cells, but a similar glutamate dehydrogenase (GDH) activity, β Cells which survive exposure to 2 mM streptozotocin only contain 10 percent of the GAD activity of control cells, but their GDH activity remains unaltered. Culture of streptozotocin‐treated β cell preparations with 2 mM nicotinamide reduces the number of dead cells and prevents in part the decline in GAD activity of surviving β cells. These data indicate that loss in activity of the β cell specific enzyme GAD can serve as marker for β cells which survived a destructive process. It is furthermore demonstrated that nicotinamide increases the percent surviving cells and decreases their loss in GAD activity.
Interaction of mutagenic tryptophan pyrolysate with DNA
Authors: Inohara Takeo , Tarui Mariko , Doi Mitsunobu , Inoue Masatoshi , Ishida Toshimasa
Published: July 2002

The interactions of DNA duplexes with 3‐amino 1,4‐dimethyl‐5H‐pyrido[4,3‐b]indole(Trp‐P‐1), a potent mutacarcinogen isolated from tryptophan pyrolysate, have been studied using CD spectroscopy. The results are that (a) the spectral change of B‐form DNA caused by the interaction with Trp‐P‐1 is biphasic, i.e. the enlargement of CD bands characteristic to the B‐DNA conformation in the range of r ([Trp‐P‐1]/[DNA]) = 0–2.5, followed by the rapid transition to the non‐B conformation at r > 2.5; (b) this transition degree of B to non‐B conformation of DNA is not necessarily dependent on the G‐C content; and (c) the salt‐induced Z‐DNA is transformed to B‐DNA (0 < r < 0.1) and then to non‐B‐DNA (r > 5), depending on the concentration of Trp‐P‐1 added. These data indicate that the non‐covalent interaction of Trp‐P‐1 with DNA is mainly dependent on the B‐DNA conformation.
The C‐terminal region of the S component of Staphylococcal leukocidin is essential for the biological activity of the toxin
Authors: Nariya Hirofumi , Izaki Kazuo , Kamio Yoshiyuki
Published: July 2002

The Staphylococcal toxin leukocidin consists of two protein components, F and S. From a culture medium of Staphylococcus aureus RIMD 310925, we isolated a truncated form of S (LS2), of which the C‐terminal 17‐residue segment is missing. Unlike intact S, LS2 showed neither leukocytolytic activity in the presence of F nor affinity for monosialoganglioside GM1 (GM1). When excited at 280 nm, both S and LS2 exhibited intrinsic tryptophan fluorescence with an emission maximum at 318 nm. Upon binding to GM1, the emission maximum of S underwent a blue shift to 310 nm, whereas no change in fluorescence took place on mixing GM1 with LS2. We conclude that the C‐terminal region of S is essential for its biological activity as well as for its binding to GM1 and that this binding is accompanied by a conformational change of the S protein.