bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of the chloroplast ATP synthetase with the photoreactive nucleotide 3′‐O‐(4‐benzoyl)benzoyl adenosine 5'‐diphosphate
Authors: Bar-Zvi Dudy , Tiefert Marjorie A. , Shavit Noun
Published: July 2002

The photoreactive nucleotide 3'‐O‐(4‐benzoyl)benzoyl ADP (BzADP) is not a substrate for photophosphorylation but is a strong competitive inhibitor (K
i 2‐25μM) with respect to ADP and ATP in photophosphorylation or ATP hydrolysis and Pi‐ATP exchange reactions, respectively. The analog binds tightly to the membrane‐bound CF1, competes with the right binding of ADP, and prevents the inactivation of the enzyme by tight binding of ADP. Upon irradiation with long wavelength ultraviolet light, the tightly bound BzADP becomes covalently attached to both the α‐ and β‐subunits of the enzyme.
Phosphorylation sites for ribosomal S6 protein kinases in mouse 3T3 fibroblasts stimulated with platelet‐derived growth factor
Authors: Wettenhall Richard E.H. , Chesterman Colin N. , Walker Tricia , Morgan Francis J.
Published: July 2002

Platelet release products and purified platelet‐derived growth factor stimulated the phosphorylation of ribosomal protein S6 in cultured mouse Balb/c 3T3 fibroblasts. The post‐nuclear fraction of the stimulated cells was enriched in S6 kinase activity specific for sites resembling those phosphorylated within intact cells in response to PDGF as determined by tryptic peptide mapping. 3T3‐S6 sites closely resembled those phosphorylated in S6 of rat hepatocytes stimulated with insulin and included sites for both cAMP‐dependent and independent kinases.
The production of hydroxyl radicals by adriamycin in red blood cells
Authors: Bannister J.V. , Thornalley P.J.
Published: July 2002

Spin trapping of the free radicals formed from the interaction between adriamycin and red blood cells resulted in the formation of a hydroxyl spin adduct. The formation of hydroxyl radicals was found to be inhibited by mannitol. Hemoglobin was found not to be obligatory for the formation of hydroxyl radicals which probably result from the reduction of hydrogen peroxide by adriamycin semiquinone.
A mechanism for the transfer of the carboxyl‐group from 1'‐N‐carboxybiotin to acceptor substrates by biotin‐containing enzymes
Authors: Goodall G.J. , Prager R. , Wallace J.C. , Keech D.B.
Published: July 2002

Previous proposals for the mechanism by which biotin‐dependent enzymes catalyse the transfer of the carboxyl group from 1'‐N‐carboxybiotin to acceptor molecules do not appear to be consistent with all of the experimental observations now available. We propose a multi‐step mechanism in which (a) substrate and then carboxybiotin bind at the second partial reaction site, (b) a base positioned adjacent to the 3'‐N of the carboxybiotin abstracts a proton from the 3'‐N and (c) the resulting enolate ion and the acceptor substrate undergo a concerted reaction resulting in carboxyl‐group transfer.
Reconstitution of solubilised brain D2 dopamine receptors into phospholipid vesicles
Authors: Wheatley M. , Strange P.G.
Published: July 2002

D2 dopamine receptors have been solubilised from bovine caudate nucleus using cholate/sodium chloride in the presence of soyabean phospholipid. Reconstitution of the receptors into soyabean phospholipid vesicles has been achieved by dialysis to remove detergent and salt. The receptors are truly reconstituted as judged by sedimentation, electron microscopy, heat stability and analysis on sucrose density gradients. The ligand‐binding properties of the reconstituted receptors resemble those of the solubilised preparation.
Polyphosphoinositide hydrolysis by phospholipase C is accelerated by thyrotropin releasing hormone (TRH) in clonal rat pituitary cells (GH3 cells)
Authors: Schlegel Werner , Roduit Claude , Zahnd Gaston R.
Published: July 2002

Thyrotropin releasing hormone (TRH) accelerates the turnover of phosphatidylinositol in GH3 cells (‘phospholipid response’). From the analysis of inositol phosphates in the presence of Li+ which inhibits their dephosphorylation, it can be concluded that the hydrolysis of phosphatidylinositol 4,5‐biphosphate, and possibly of phosphatidylinositol 4‐phosphate by phospholipase C is markedly accelerated by TRH. It appears that this reaction initiates the acceleration of phosphatidylinositol turnover. The specificity of hormonally regulated phospholipase C reaction for polyphosphoinositides has important implications for the potential role of the phospholipid response as a mechanism of membrane signal transduction.
Potentiation of actomyosin ATPase activity by filamin
Authors: Sosiński J. , Szpacenko A. , Dabrowska Renata
Published: July 2002

It was found that thin filaments from chicken gizzard muscle activate skeletal muscle myosin Mg2+‐ATPase to a greater extent than does the complex of chicken gizzard actin and tropomyosin. The protein factor responsible for this additional activation has been now identified as the high M
r actin binding protein, filamin.
Activation of plant aspartate kinase by calcium and calmodulin‐like factor from plants
Authors: Sane P.V. , Kochhar S. , Kumar Nikhil , Kochhar V.K.
Published: July 2002

Aspartate kinase partially purified from plants is activated by a protein factor prepared from the same plant material. This activation is further stimulated by calcium and inhibited by anticalmodulin compounds such as trifluoperazine and chlorpromazine. The results suggest that the characteristics of regulation of the enzyme activity by the protein factor are similar to calmodulin regulation of plant and animal enzymes.
Transformation of leukotriene A4 methyl ester to leukotriene C4 monomethyl ester by cytosolic rat glutathione transferases
Authors: Mannervik Bengt , Jensson Helgi , Ålin Per , Örning Lars , Hammarström Sven
Published: July 2002

Six major basic cytosolic glutathione transferases from rat liver catalyzed the conversion of leukotriene A4 methyl ester to the corresponding leukotriene C4 monomethyl ester. Glutathione transferasc 4‐4, the most active among these enzymes, had a V
maxof 615 nmol · min−1 · mg protein−1 at 30°C in the presence of 5 mM glutathione. It was followed in efficiency by transferase 3–4 which had a V
max of 160 nmol · min−1 · mg−1 under the same conditions. Transferases 1‐1, 1‐2, 2‐2 and 3‐3 had at least 30 times lower V
max values than transferase 4‐4.
Formation of leukotriene C4 by human leukocytes exposed to monosodium urate crystals
Authors: Serhan Charles N. , Lundberg Ulf , Lindgren Jan Åke , Weissmann Gerald , Samuelsson Bengt
Published: July 2002

Monosodium urate (MSU) crystals stimulate the metabolism of arachidonic acid in mixed populations of human leukocytes. Leukocytes exposed to MSU crystals released leukotriene C4. Leukotriene C4 (LTC4) was characterized and detected by high‐performance liquid chromatography (HPLC), UV absorption, bioassay with guinea pig ileum, and radioimmunoassay. Results indicate that MSU crystals stimulate the transformation of arachidonic acid and the formation of leukotriene C4 in human leukocytes; an effect inhibited by colchicine. Moreover, they suggest that LTC4 may serve as a mediator of inflammation in crystal‐associated diseases.
The anomalous effect of some ACTH‐fragments missing the amino acid sequence 1–10 on the corticosteroidogenesis in purified isolated rat adrenal cells
Authors: Goverde H.J.M. , Smals A.G.H.
Published: July 2002

The corticosteroidogenicity of ACTH‐derived peptides was tested in a purified isolated rat adrenal cell system. The activity was related to the activity of standard ACTH (synthetic hACTH1–39; potency fixed at 100). The peptides ACTH11–19NH2 and ACTH11–24 showed steroidogenicity at pharmacological doses (potencies: 0.00067 and 0.00032, respectively). None of the peptides tested inhibited or potentiated the ACTH‐induced steroidogenesis at low doses (0.5–50 000 pg). The results suggest the presence of a second center within the ACTH molecule capable of inducing steroidogenesis.
Photoaffinity labeling of the coupling factor 1 from the thermophilic bacterum PS3 by 8‐azido ATP
Authors: Schäfer Hans-Jochen , Scheurich Peter , Rathgeber Gabriele , Dose Klaus , Kagawa Yasuo
Published: July 2002

To localize the nucleotide binding sites of the F1ATPase (TF1) from the thermophilic bacterium PS3 we have used 14C‐labeled 8‐azido ATP (8‐N3ATP) as photoaffmity label. 8‐N3ATP is hydrolyzed by the F,ATPase in the absence of ultraviolet light. Irradiation by ultraviolet light of the enzyme in the presence of 8‐N3ATP results in reduction of ATPase activity and in preferential nucleotide specific labeling of the α subunits (0.8–0.9 mol 8‐N3ATP/TF1,α:β = 4:1). Inactivation and labeling do not depend on the presence of Mg2+. Both effects decrease upon addition of various nucleotide di‐ or triphosphates.
S‐[18F]Acetylcyclofoxy: a useful probe for the visualization of opiate receptors in living animals
Authors: Pert Candace B. , Danks Janine A. , Channing Michael A. , Eckelman William C. , Larson Steven M. , Bennett Jean M. , Burke Terrence R. , Rice Kenner C.
Published: July 2002

Opiate receptor
PET
3‐[18F]Acetylcyclofoxy
Stereospecificity
Naloxone
Evidence for the phosphorylation of enzyme IIglucose of the phosphoenolpyruvate—sugar phosphotransferase system of Escherichia coli and Salmonella typhimurium
Authors: Peri Krishna G. , Hans L Kornberg , Waygood E.Bruce
Published: July 2002

A membrane‐bound phosphoprotein, of subunit Mr 48 000 by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, has been found in Escherichia coli and Salmonella typhimurium. The phosphorylation of this protein is dependent upon phosphoenolpyruvate and the protein of the sugar phosphotransferase (PT) system: enzyme I, HPr, and IIIglucose. The membrane‐bound phosphoprotein is identified as enzyme IIglucose. Other membrane‐bound phosphoproteins, that are also dependent upon the PT system, remain to be identified.
NADPH oxidase of neutrophils forms superoxide anion but does not reduce cytochrome c and dichlorophenolindophenol
Authors: Bellavite Paolo , della Bianca Vittorina , Serra M.Cristina , Papini Emanuele , Rossi Filippo
Published: July 2002

Superoxide (O−
2) production by partially purified NADPH oxidase from guinea pig neutrophils was markedly increased when the cells were activated by exposure to phorbol‐myristate acetate. On the contrary, NADPH‐dependent cytochrome c and 2,6‐dichlorophenolindophenol (DCIP) reductase activities in preparations from resting and activated neutrophils were similar. The apparent K
m values for NADH and NADPH of the reductase activities were different from those of the O−
2 producing enzyme. The electron acceptors did not inhibit the oxygen consumption by NADPH oxidase in the presence of superoxide dismutase. Even in anaerobiosis the oxidase failed to reduce cytochrome c and DCIP. These results suggest that NAD(P)H‐dependent dye reductase activities are not involved in the electron transport system responsible for the O−
2 production by neutrophils.
Monoclonal antibodies localize the exchangeable GTP‐binding site in β‐ and not α‐tubulins
Authors: Hesse Juliane , Maruta Hiroshi , Isenberg Gerhard
Published: July 2002

A combination of several methods was used to localize the exchangeable GTP‐binding site in the α‐/β‐tubulin heterodimer: (i) direct photoaffinity labeling with [α‐32P]GTP, (ii) specific labeling of α‐ and β‐tubulin by tyrosylation and phosphorylation, respectively, and (iii) immunoprecipitation with specific monoclonal antibodies. Direct evidence was obtained that GTP binds exclusively to β‐ and not α‐tubulins.
23Na NMR investigation of human lenses from patients with cataracts
Authors: Fossarello Maurizio , Orzalesi Nicola , Corongiu Francesco P. , Biagini Simona , Casu Mariano , Lai Adolfo
Published: July 2002

23Na NMR
Lens
Cataract
Molecular mechanisms regulating the synthesis of transferrin receptors and ferritin in human erythroleukemic cell lines
Authors: Louache F. , Pelosi E. , Titeux M. , Peschle C. , Testa U.
Published: July 2002

The effects of either iron salts or iron chelators on the biosynthesis of transferrin receptors in human erythroleukemic lines was investigated. Addition of these compounds induced a rapid and marked decrease (iron salts) or increase (iron chelators) in the level of transferrin receptors synthesis. Both phenomena were inhibited by actinomycin D. In contrast, the increase in the synthesis of ferritin induced in these cells by addition of iron salts was not inhibited by actinomycin D. These results suggest that iron salts modulate the synthesis of transferrin receptors and ferritin via different molecular mechanisms, of transcriptional and translation type, respectively.
Localization of the continuous allergenic sites of ragweed allergen Ra3 by a comprehensive synthetic strategy
Authors: Atassi Hammad , Atassi M.Zouhair
Published: July 2002

A comprehensive synthetic approach, previously introduced by this laboratory for the localization of the full profile of the continuous antigenic sites on proteins, was applied here to localize the continuous sites of ragweed allergen, Ra3, that are recognized by human anti‐Ra3 IgE antibodies. The following 10 uniform and overlapping peptides were synthesized and purified: 1–15, 11–25, 21–35, 31–45, 41–55, 51–65, 61–75, 71–85, 81–95 and 91–101. Quantitative radiometric titrations of protein and peptide adsorbents with human IgE, established the full profile of allergenic (IgE binding) sites on Ra3. It was found that Ra3 has four continuous allergenic sites. Antibodies prepared against the IgE binding peptides bound to native Ra3. The findings are briefly discussed in relation to other protein antigenic structures and in terms of design of vaccines using synthetic sites.
An examination of the ability ofinositol 1,4,5‐trisphosphate to induce calcium release and tension development in skinned skeletal muscle fibres of frog and crustacea
Authors: Lea T.J. , Griffiths P.J. , Tregear R.T. , Ashley C.C.
Published: July 2002

We have examined the ability of inositol 1,4,5‐trisphosphate (InsP3) to cause contractions of mechanically skinned muscle fibres of frog and barnacle. InsP3 (10–500 μM) did not cause any tension development in 25 frog skinned fibres and 26 barnacle myofibrillar bundles, although contractions could be readily evoked by caffeine and by replacement of an impenneant anion by Cl−, treatments known to release calcium from the sarcoplasmic reticulum (SR). Four barnacle bundles did give responses to InsP3. InsP3 did not modify responses to caffeine or calcium‐induced calcium release. Free Mg2+ was lowered to 40 μM and 15 mM D‐2,3‐diphosphoglycerate was added in order to inhibit the possible breakdown of InsP3 by inositol trisphosphatase. Neither measure revealed a response to InsP3. Arsenazo III absorbance measurements failed to detect any binding of Mg2+ (0–0.5 mM) by 0.35 mM InsP3 in our solutions. Inhibitors of SR calcium uptake (cadmium, quercetin, furosemide), omission of EGTA from the solution and varying the temperature from 4° to 22°C also failed to reveal a response of frog skinned fibres to InsP3. The nucleotide GTP, which has been reported to enhance InsP3‐induced calcium release from rat liver microsomes, had no effect at 50 μM on the response of frog fibres to InsP3. It is concluded that under conditions in which other calcium release mechanisms operate well, InsP3 is relatively ineffective at releasing calcium from the SR in amounts sufficient to induce contraction. Although we have been unable to find evidence to support the proposed role of InsP3 as an essential link in excitation‐contraction coupling of skeletal muscle, we cannot entirely reject its role if essential cofactors are lost in the skinned preparations.
Autoradiographic localisation of VIP receptors in human lung
Authors: Leys K. , Morice A.H. , Madonna O. , Sever P.S.
Published: July 2002

Localisation and pharmacological properties of the VIP receptor in human lung sections are described. The receptor density determined by saturation analysis using 125I‐VIP is approx. 100 protein, with a k
d of approx. 600 pM. Inhibition of 125I‐VIP binding with VIP and related peptides gives a rank order of potency: VIP /s> peptide histidine methionine /s> secretin. Light microscope autoradiography reveals specific VIP binding sites, with a high density over the pulmonary artery smooth muscle and the alveolar walls and with a lower density over the bronchial epithelium.
Exon structure of the collagen‐binding domain of human fibronectin
Authors: Owens Raymond J. , Baralle Francisco E.
Published: July 2002

Fibronectin consists of a series of three internal homology repeats (types I, II, and III [(1983) Proc. Natl. Acad. Sci. USA 80, 137‐141]). The type III units are each coded for by two exons with the exception of the alternatively spliced extra domain type III [(1984) EMBO J. 3, 2511‐2516]. To investigate the gene organisation of the type I and II repeats, genomic clones covering the collagen‐binding domain of human fibronectin have been isolated and characterised. The results show that each of the type I and II homology units in this region corresponds to an exon. Taken together with the previous data concerning the type III units, the data lend support to a gene fusion model of fibronectin evolution.
Recombinant human B cell stimulatory factor 2 (BSF‐2/IFN‐β2) regulates β‐fibrinogen and albumin mRNA levels in Fao‐9 cells
Authors: Andus Tilo , Geiger Thomas , Hirano Toshio , Northoff Hinnak , Ganter Ursula , Bauer Joachim , Kishimoto Tadamitsu , Heinrich Peter C.
Published: July 2002

Conditioned medium from human monocytes contains a partially characterized hepatocyte‐stimulating factor that simultaneously elevates the mRNA levels of the acute‐phase protein β‐fibrinogen and decreases albumin mRNA in rat hepatoma cells. We demonstrate that recombinant human B‐cell stimulatory factor 2, which is identical to interferon‐β2/26 kDa protein and interleukin‐HP1, exhibits the same activity as hepatocyte‐stimulating factor. Furthermore, a specific antibody against B‐cell stimulatory factor 2 was able to inhibit hepatocyte‐stimulating factor in conditioned medium from human monocytes. Our data show that hepatocyte‐stimulating factor and B‐cell stimulatory factor 2 are functionally and immunologically related proteins.
Purification and subunit structure of hepatocyte growth factor from rat platelets
Authors: Nakamura Toshikazu , Nawa Katsuhiko , Ichihara Akira , Kaise Nobuko , Nishino Tomoyoshi
Published: July 2002

A hepatocyte growth factor (HGF) that stimulates DNA synthesis of adult rat hepatocytes in primary culture was purified as a homogeneous material from platelets of 1000 rats by a four‐step procedure: stimulation of its release from platelets by thrombin, cation‐exchanger fast protein liquid chromatography (FPLC) on a Mono S column, heparin‐Sepharose CL‐6B chromatography, and reverse‐phase HPLC on a C4 column. The purified HGF stimulated DNA synthesis of adult rat hepatocytes in primary culture at 1 ng/ml and was maximally effective at 5 ng/ml, being about twice as potent as EGF at this concentration. HGF did not stimulate DNA synthesis of Swiss 3T3 cells. It was found to be a beat‐ and acid‐labile protein that was inactivated by reduction with dithiothreitol. The purified HGF had a molecular mass of 82 kDa, as estimated by SDS‐PAGE, and was found to be a heterodimer which dissociated into a large subunit of 69 kDa and a small one of 34 kDa by SDS‐PAGE under reducing conditions. These biological and chemical properties showed that HGF was not identical with any known growth factors, including platelet‐derived growth factor (PDGF).
Variations with position of replication errors due to exonuclease warm‐up
Authors: Lecomte Philippe J. , Ninio Jacques
Published: July 2002

A.A mismatch errors occuring during poly(dA) replication with the Klenow fragment of E. coli DNA polymerase I have been qualified. The A/T ratio measured for chains extended by 1–25 nucleotides decreases by a factor of at least 15 from beginning to end. The deduced true error rate may decrease by a factor of 2.5 at each successive nucleotide addition. When ddATP is used instead of dATP, the ddA/T ratio indicates little variation of the misincorporation probability with position. Thus, the accuracy improvement in the first case is due to a warm‐up of the proofreading function.
Effect of inositol 1,4,5‐trisphosphate and GTP on calcium release from pituitary microsomes
Authors: Kiesel L. , Lukács G.L. , Eberhardt Inge , Runnebaum B. , Spät A.
Published: July 2002

Microsomal vesicles from bovine anterior pituitary accumulate Ca2+ and maintain a steady‐state ambient Ca2+ level of 200 nM. IP3 and GTP both induce calcium release from the microsomal vesicles. The effect of IP3 is inhibited by polyethylene glycol (PEG), and the effect of GTP is absolutely dependent on PEG. Half‐maximal effect of IP3 (without PEG) is 0.26 μm, the maximal calcium release attaining 7% of the A23187‐releasable pool. The same values for GTP (in the presence of PEG) are 80 μM and 10%, respectively. GTP potentiates the effect of IP3. This potentiation is not mediated by protein phosphorylation.
Nuclear matrix and chromosome scaffold preparations of in vitro cultured bovine liver cells have two proteins in common
Authors: Pieck Anneke C.M. , Rijken Antonia A.M. , Wanka Friedrich
Published: July 2002

Nuclear matrices and chromosome scaffolds of in vitro cultured bovine liver cells were prepared under conditions that preserve the specific binding of the DNA. Protein compositions were analysed by electrophoresis and peptide mapping. Two slightly acidic polypeptides of apparent molecular masses 47 and 53 kDa were present in nuclear matrix as well as chromosome scaffold preparations. The corresponding matrix and scaffold proteins had identical peptide maps. Their putative function in the spatial organization of the DNA during the cell cycle is considered.
Complement‐mediated adherence of immune complexes to human erythrocytes
Authors: Schifferli Jurg A. , Hauptmann Georges , Paccaud Jean-Pierre
Published: July 2002

The classical pathway of complement is required for the adherence of soluble tetanus toxoid (TT)‐human anti‐TT complexes to erythrocytes. Using human C4‐deficient serum we compared the capacity of the two forms of human C4 (C4A and C4B) to mediate this function: C4A was shown to be 1.5‐fold more efficient than C4B. In contrast, haemolysis by C4B was 3.7‐fold more efficient than by C4A. Such large differences suggest that both forms are complementary, and that C4A is preferentially involved in the processing of immune complexes in humans.
Adenosine decreases intracellular free calcium concentrations in cultured vascular smooth muscle cells from rat aorta
Authors: Kai Hisashi , Kanaide Hideo , Matsumoto Takahiro , Shogakiuchi Yoshito , Nakamura Motoomi
Published: July 2002

Using an intracellularly trapped dye, quin 2, effects of adenosine on intracellular free calcium concentrations ([Ca2+]i) were recorded, microfluorometrically, using rat aortic medial vascular smooth muscle cells (VSMCs) in primary culture. Regardless of whether cells were at rest (in 5 mM K+), at K+‐depolarization (in 55 mM K+) or at Ca2+ depletion (in Ca2+‐free media), adenosine induced a rapid reduction of [Ca2+]i, following which there was a gradual increase to pre‐exposure levels, in cells at rest and in the case of Ca2+ depletion. Only when the cells were depolarized (55 mM K+) did adenosine induce a new steady [Ca2+]i level, lower than the pre‐exposure value. These findings indicate that decrease in [Ca2+]i by adenosine is one possible mechanism involved in the adenosine‐mediated vasodilatation, and that adenosine decreases [Ca2+]i by direct extrusion, by sequestration, or by inhibiting the influx of Ca2+ into VSMCs.
Brush border myosin heavy chain phosphorylation is regulated by calcium and calmodulin
Authors: Rieker James P. , Swanljung-Collins Helena , Montibeller Judith , Collins Jimmy H.
Published: July 2002

Myosin from chicken intestinal brush borders is phosphorylated on its heavy chains at threonine by a kinase isolated from brush borders. In contrast to other heavy chain kinases, the brush border kinase activity is dependent on calcium and calmodulin. The partially purified preparation also phosphorylated myosin on its light chains at serine, but in a calmodulin‐independent manner. Phosphorylation of the light chains in the absence of calmodulin or both heavy and light chains in the presence of calmodulin activated its actinactivated ATPase activity about 10‐fold, to about 50 per mg.
Functional multiplicity of atrial natriuretic peptide receptors on cultured rat Leydig tumor cells
Authors: Vlasuk George P. , Arcuri Karen E. , Ciccarone Terry M. , Nutt Ruth F.
Published: July 2002

Native rat atrial natriuretic peptide (NANP) was shown to bind with high affinity and to increase intracellular levels of cGMP in cultured rat Leydig tumor cells. A linear analog of NANP which lacks the disulfide‐linked bridge structure also bound with high affinity but did not increase levels of intracellular cGMP or antagonize the increase of this cyclic nucleotide by NANP. These data are consistent with the existence of two functional subpopulations of ANP receptors on cultured rat Leydig tumor cells; one which is capable of activating guanylate cyclase and one which is not linked to this enzyme.
Carbon‐phosphorus lyase activity in permeabilized cells of Arthrobacter sp. GLP‐1
Authors: Pipke Rüdiger , Amrhein Nikolaus
Published: July 2002

Arthrobacter sp. GLP‐1 is capable of using the phosphonate herbicide glyphosate (N‐[phosphonomethyl]glycine) as its sole source of phosphorus. The formation of sarcosine from glyphosate by a carbon‐phosphorus (CP) lyase activity could be demonstrated in vivo, as well as in situ in cells permeabilized by 2% dimethylsulfoxide, when sarcosine oxidase was inhibited by acetate. The apparent K
m of the CP lyase for glyphosate was found to be 61 μM, and a V of 2.2 pmol · s−1 · mg−1 soluble protein was determined. Uptake of glyphosate into the cells and CP lyase activity represent distinct processes.
Phosvitin kinase activity in Acholeplasma axanthum
Authors: Platt Mark W. , Rottem Shlomo
Published: July 2002

Incubating the soluble fraction derived from A. axanthum with phosvitin and [γ‐32P]ATP results in the phosphorylation of phosvitin. Casein, histone and kemptide were practically ineffective substrates, whereas a 55 kDa protein of M. gallisepticum was efficiently phosphorylated. The enzymatic activity has an optimal pH in the pH range 6.0–6.2 and requires divalent cations. The activity was inhibited by ammonium sulfate, heparin and sulphydryl blocking reagents, but was not affected by calmodulin with or without Ca2+ or by cyclic AMP.
Retinoic acid inhibits phospholipid turnover and protein kinase C activity in RA‐sensitive but not in RA‐resistant cells
Authors: Vanier M.C. , Banerjee D. , Mukherjee B.B.
Published: July 2002

Treatment with 10−5 M retinoic acid causes loss of anchorage‐independent growth in src‐transformed RR1022 cells but not in ras‐transformed KNRK cells. In an effort to elucidate the mechanisms underlying this difference, we investigated the effect of RA on phospholipid turnover and PKC activity in these two cell lines. 10−5 M RA treatment caused a drastic inhibition of 32P incorporation into PI and PA and a large increase in 32P incorporation into PC in RR1022 cells. Similar treatment of KNRK cells yielded no change in PC or PA labelling and a much smaller decrease in PI labelling. Furthermore, 10−5 M RA treatment causes a large decrease in PKC activity in RR1022 cells (35% of control) but only a small decrease in KNRK cells (78% of control). We suggest that these effects are part of an altered signal transduction pathway which mediates the differential effects of RA on anchorage‐independent growth in these two cell lines.
Insulin and progesterone activate a common synthetic ribosomal protein S6 peptide kinase in Xenopus oocytes
Authors: Cicirelli Michael F. , Pelech Steven L. , Krebs Edwin G.
Published: July 2002

A synthetic peptide Arg‐Arg‐Leu‐Ser‐Ser‐Leu‐Arg‐Ala, the structure of which is based on that of a phosphorylated sequence in ribosomal protein S6, was employed as a probe for stimulated kinase activity in Xenopus laevis oocytes induced to mature with insulin or progesterone. Insulin elicited an early (20–30 min) 3‐fold stimulation of S6 peptide phosphorylating activity that was not evident with progesterone. However, both hormones produced a delayed 7–12‐fold stimulation of S6 peptide phosphorylating activity at the time of germinal vesicle breakdown. The results of DEAE‐Sephacel, Sephacryl S‐200, TSK‐400, and heparin‐Sepharose chromatographic fractionation experiments imply that a common S6 peptide kinase is activated as a consequence of short and long term insulin exposure, as well as in long term progesterone treatment of oocytes. Omission of potassium from the oocyte culture medium greatly facilitated insulin‐induced meiotic maturation.
NAD+ stimulated the spermidine‐dependent hypusine formation on the 18 kDa protein in cytosolic lysates derived from NB‐15 mouse neuroblastoma cells
Authors: Chen Kuang Yu , Dou Qing Ping
Published: July 2002

When incubated with cultured mouse neuroblastoma cells under growth stimulatory condition, [3H]putrescine or [3H]spermidine can metabolically label a cellular protein of apparent molecular mass 18 kDa. The labeling, which leads to hypusine formation, is due to a covalent linkage between a lysine residue and the butylamino group derived from spermidine. This reaction can be demonstrated in the cytosolic fractions obtained from cells whose spermidine pool was depleted by prior treatment with α‐difluoromethylornithine. In an effort to characterize the enzyme system involved in this unique post‐translational modification, we found that NAD+ at 0.1 mM stimulated labeling more than 150‐fold. Other nucleotides such as NADP+, ATP and GTP were ineffective. The fact that NAD+ dramatically stimulated labeling of the 18 kDa protein indicated that the enzyme involved in hypusine formation may be an NAD+‐requiring enzyme.
Different sensitivities of the responses of human neutrophils stimulated with immune complex and C5a anaphylatoxin to pertussis toxin
Authors: Shirato Manabu , Takahashi Kazuhiko , Nagasawa Shigeharu , Koyama Jiro
Published: July 2002

When stimulated with immune complex or C5a anaphylatoxin, human neutrophils undergo an increase in the concentration of intracellular Ca2+ ([Ca2+]i) that precedes the onset of superoxide (O−
2) production. The extracellular Ca2+ is required for the O−
2 production of neutrophils stimulated by C5a, but is only partially required for those by immune complex. The addition of pertussis toxin to neutrophils does not inhibit the rise in [Ca2+]i and O−
2 production induced by immune complex but does inhibit those induced by C5a. These results suggest that a different sequence of reaction is involved by different stimulants, the anaphylatoxin and immune complex.
Trapping of transiently formed quinone methide during enzymatic conversion of N‐acetyldopamine to N‐acetylnorepinephrine
Authors: Sugumaran Manickam , Saul Steven , Semensi Victor
Published: July 2002

We have demonstrated that quinone methide formation is an important aspect of insect physiology and proposed that enzymatically generated quinone methides react nonenzymatically with water or other nucleophiles to form Michael‐1,6‐addition products [(1988) Adv. Insect Physiol. 21, 179–231; (1989) J. Cell. Biochem. suppl. 13C, 58]. Using a purified o‐quinone isomerase from the larval cuticle of Sacrophaga bullata and mushroom tyrosinase, we now demonstrate that transiently formed N‐acetyldopamine quinone methide from N‐acetyldopamine can be trapped by methanol to produce β‐methoxy N‐acetyldopamine. The methanol adduct thus formed was found to be a racemic mixture and can be resolved into the optical isomers on cyclodextrin chiral column. These results confirm our contention that enzymatically generated quinone methides are nonenzymatically and nonstereoselectively transformed to Michael‐1,6‐adducts by reaction with water or other nucleophiles.
EGF‐like domains in extracellular matrix proteins: Localized signals for growth and differentiation?
Authors: Engel Jürgen
Published: July 2002

Multidomain proteins of the extracellular matrix (ECM) play an important role in development and maintenance of cellular organization and in tissue repair. Several ECM proteins such as laminin, tenascin, and thrombospondin contain domains with homology to epidermal growth factor (EGF) and exhibit growth promoting activity. The mitogenic activity of laminin is restricted to a fragment which consists of about 25 repeating domains with partial homology to EGF and comprises the rod‐like inner regions of the three short arms of the four armed molecule. The mitogenic activity does not correlate with promotion of cell attachment and neurite outgrowth for which major functional sites have been found in other regions of the laminin molecule. It is suggested that EGF‐like domains in laminin, in other ECM proteins and in the extracellular portions of some membrane proteins are signals for cellular growth and differentiation. Because they are integral parts of large molecules and often of supramolecular assemblies these domains are well suited to stimulate neighboring cells in a specific and vectorial way. This concept of localized growth or differentiation signals offers an attractive mechanism for the regulation of cellular development.
Phosphatidylinositol hydrolysis by human plasma phospholipase D
Authors: Balsinde Jesus , Mollinedo Faustino
Published: July 2002

A phospholipase D activity able to hydrolyze phosphatidylinositol has previously been described in the cytosol of human neutrophils. The experiments reported here demonstrate that this phosphatidylinositol‐hydrolyzing phospholipase D activity is also present in human plasma. This activity was assessed by free inositol release from phosphatidylinositol substrate, by phosphatidate formation and by phosphatidylethanol formation through its capacity of catalyzing a transphosphatidylation reaction. This plasma enzyme activity shows an optimum pH of 8.0 and is inhibited by EGTA.
Interaction of two brain annexins, CaBP33 and CaBP37, with membrane‐skeleton proteins
Authors: Giambanco Ileana , Pula Grazia , Bianchi Roberta , Donato Rosario
Published: July 2002

CaPB33 and CaPB37, two annexins purified from bovine brain, interact with a Triton X‐100‐resistant fraction (cytoskeleton) from bovine brain membranes in a Ca2+‐dependent way in vitro. The binding is saturable with respect to the CaBP33–CaBP37 concentration, half‐maximal binding occurring at ~15 μg of the CaBP33–CaBP37 mixture/ml. The binding of these two annexins to the crude cytoskeleton preparation as a function of free Ca2+ concentration is biphasic, with half‐maximal binding at ~ 50 μM and ~ 400 μM free Ca2+ for the first and the second component, respectively. By an overlay technique, CaBP33 and CaBP37 bind to a set of low M
r polypeptides (10–20 kDa) in the crude cytoskeleton preparation, with formation of an 85–90 kDa complex as investigated in cross‐linking experiments. No binding of the CaBP33–CaBP37 mixture to either G‐or F‐actin has been observed. Identification of the CaBP33–CaBP37‐binding proteins in cytoskeletons would help elucidating the function(s) of these annexins in the brain.
Structural features of the McPC603 Fab fragment not defined in the X‐ray structure
Authors: Skerra Arne , Glockshuber Rudi , Plückthun Andreas
Published: July 2002

The proteolytic Fab fragment of the well characterized antibody McPC603 was compared to the recombinant Fab fragment, which was obtained in functional form from an Escherichia coli expression system [(1989) Methods Enzymol. 178, 497‐515]. We found evidence that the proteolytic fragment is glycosylated at Asn H160 in the CH1 domain, where additional electron density had been observed in the crystal structure [J. Mol. Biol. 190, 593‐604]. In addition, its heavy chain is about 30 amino acids longer than visible in the electron density and thus contains the complete hinge region. These structural differences between the recombinant Fab fragment, which had been designed exactly according to the defined electron density, and the proteolytic Fab fragment of McPC603 had no effect on the hapten binding properties of these antigen binding fragments. Yet, it may be important to be aware of these structural features of McPC603 in folding studies and some comparative analyses of antibody structures.
Spin‐spin relaxation of the phosphodiester resonance in the 31P NMR spectrum of human brain
Authors: Kilby Peter M. , Allis Jonathan L. , Radda George K.
Published: July 2002

The phosphodiester peak in 31P nuclear magnetic resonance spectra of human brain in vivo is often the most prominent feature of the spectrum. We have demonstrated that this resonance exhibits bi‐exponential spin‐spin relaxation, giving relaxation times of 2 and 10 ms. We interpret this in terms of the two components which make up the peak, bilayer lipids and the small cytosolic phosphates glycerophosphoethanolamine and glycerophosphocholine. Using the relaxation times and the relative peak heights of the two components we have also been able to quantitate the concentration of the bilayer lipids as 20–40 times that of ATP.
Sub‐site preferences of the aspartic proteinase from the human immunodeficiency virus, HIV‐1
Authors: Konvalinka Jan , Strop Petr , Velek Jiri , Cerna Vera , Kostka Vladimir , Phylip Lowri H. , Richards Anthony D. , Dunn Ben M. , Kay John
Published: July 2002

A series of synthetic, chromogenic substrates for HIV‐1 proteinase with the general structure Ala‐Thr‐His‐Xaa‐Yaa‐Zaa∗Nph‐Val‐Arg‐Lys‐Ala was synthesised with a variety of residues introduced into the Xaa, Yaa and Zaa positions. Kinetics parameters for hydrolysis of each peptide by HIV‐1 proteinase at pH 4.7, 37°C and u = 1.0 M were measured spectrophotometrically and/or by reverse phase FPLC. A variety of residues was found to be acceptable in the P3, position whilst hydrophobic/aromatic residues were preferable in P1. The nature of the residue occupying the P2; position had a strong influence on k
cat (with little effect on k
m;β‐branched residues Val or Ile in this position resulted in considerably faster peptide hydrolysis than when e.g. the Leu‐containing analogue was present in P2.
Human chorionic gonadotropin α and human cytomegalovirus promoters are extremely active in the fission yeast Schizosaccharomyces pombe
Authors: Toyama Reiko , Okayama Hiroto
Published: July 2002

We have investigated the transcriptional activity of human cytomegalovirus, herpes thymidine kinase, human chorionic gonadotropin α, somatostatin, immunoglobulin κ chain, α crystallin, albumin and interferon‐β promoters in the fission yeast Schizosaccharomyces pombe. Among these, the human cytomegalovirus, human chorionic gonadotropin α, and somatostatin promoters were found to be very active, approximately 11‐, 9‐, and 0.9‐fold as active as the SV40 early promoter, respectively. The remainder of the promoters studied were weak, having only 10–20% of the SV40 promoter activity. Primer extension analysis showed that the strong promoters initiated transcription in S. pombe at the same sites as in mammalian cells, indicating the high similarity between both transcriptional systems.
The calcium binding sites in human annexin V by crystal structure analysis at 2.0 A resolution Implications for membrane binding and calcium channel activity
Authors: Huber Robert , Schneider Monika , Mayr Irmgard , Römisch Jürgen , Paques Eric-P.
Published: July 2002

Crystal structure analysis and refinement at 2.0 A resolution of a rhombohedral crystal form of human annexin V at high calcium concentration revealed a domain motion compared to the previously analysed hexagonal crystal form. Five calcium ions were located on the convex face of the molecule. Three strongly bound calciums are liganded at protruding interhelical loops and Asp or Glu residues in homologous positions in repeats I, II and IV. Five proteinaceous oxygens and one solvent molecule form the coordination polyhedron in each case. The unoccupied seventh site is suggested as the phospholipid headgroup binding site. Two more weakly bound sites were identified by lanthanum labelling. The structural features suggest that annexin V attaches with its convex face to membranes by specified calcium mediated interactions with at least three phospholipids. The adjacent membrane bilayer may thus become locally disordered and permeable to allow calcium inflow through the central polar channel of the molecule.
Inhibitory effect of calcium‐binding protein regucalcin on Ca2+‐activated DNA fragmentation in rat liver nuclei
Authors: Yamaguchi Masayoshi , Sakurai Takashi
Published: July 2002

Incubation of isolated rat liver nuclei with ATP, NAD+, and micromolar Ca2+ concentrations of various metal ions resulted in extensive DNA hydrolysis. Half‐maximal activity occurred with 1.0 μM Ca2+ added, and saturation of the process was observed with 10 μM Ca2+, The Ca2+ (10 μM)‐activated DNA fragmentation was inhibited by the presence of Ca2+‐binding protein regucalcin isolated from rat liver cytosol. The inhibitory effect of regucalcin was complete at 0.5 μM. At 25μM Ca2+ added, such an effect of regucalcin (1.0μM) was not seen, Regucalcin also inhibited Ca2+‐activated DNA fragmentation in the presence or calmodulin (10 and 20 μg). The results show that regucalcin can inhibit the Ca2+‐activated DNA fragmentation due to binding the metal, suggesting a role in regulation of liver nuclear functions.
AMPA and kainate‐operated channels reconstituted in artificial bilayers
Authors: Ambrosini A. , Barnard E.A. , Prestipino G.
Published: July 2002

Catlonic channels which can be activated by α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) and kainate play a key role in the generation of excitatory posisynaptic potentials in the brain of all vertebrates. On a protein carrying binding sites for both these ligands, affinity‐purified from the Xenopus nervous system, electrical measurements have been performed to investigate its functional properties after the pure complex had been incorporated into planar lipid bilayers. Domoate, AMPA or kainate added to the reconstituted protein activated cationic channels, which were blocked by typical antagonists for this neurotransmitter system. These data suggest that the reconstituted protein is an ionotropic receptor of the unitary non‐NMDA sybtype.
The inhibitors thapsigargin and 2,5‐di(tert‐butyl)‐1,4‐benzohydroquinone favour the E2 form of the Ca2+, Mg2+‐ATPase
Authors: Wictome Matthew , Michelangeli Francesco , Lee Anthony G. , East J.Malcolm
Published: July 2002

2,5‐Di(tert‐butyl)‐1,4‐benzohydroquinone has been shown to inhibit the Ca2+, Mg2+‐ATPase of sarcoplasmic reticulum with an affinity of 0.4 μM. It has been shown to shift the E2‐E1 equilibrium for the ATPase towards E2, as shown previously for the inhibitor thapsigargin. The shift towards E2 results in a decrease in affinity for Ca2+, as also observed for thapsigargin. A marked decrease in the rate of the E2‐E1 transition is observed for both BHQ and thapsigargin. A decrease in the equilibrium level of phosphorylation by Pi and of the steady‐state lever of phosphorylation by ATP are consistent with a decrease in the equilibrium constant for phosphorylation by Pi and an increase in the rate of dephosphorylation.
Crosslinking of nuclear proteins to DNA by cis‐diammminedichloroplatinum in intact cells Involvement of nuclear matrix proteins
Authors: Ferraro Anna , Grandi Paolo , Eufemi Margherita , Altieri Fabio , Turano Carlo
Published: July 2002

In order to detect the nuclear matrix proteins involved in DNA binding, avoiding possible artifacts derived from the disruption of nuclei, proteins were crosslinked to DNA by the action of cis‐diamminedichloroplatinum on intact chicken liver cells and analyzed by two‐dimensional gel electrophoresis. At least eleven species of crosslinked proteins were found to derive from the nuclear matrix prepared from the same cell type, and five of these were found also among the proteins crosslinked to DNA in intact liver cells from ox and pig. This subset of common proteins, conserved in different animal species, is likely to have a fundamental role for the anchorage of DNA to the nuclear matrix.
The Escherichia coli cysG gene encodes the multifunctional protein, siroheme synthase
Authors: Spencer Jonathan B. , Stolowich Neal J. , Roessner Charles A. , Scott A.Ian
Published: July 2002

Previously, the E. coli cysG gene product had been shown to sequentially methylate uro'gen III to produce precorrin‐2, hence it was given the trivial name uro'gen III methylase. We now report that in addition to methylase activity, the CysG protein catalyses both the NAD+ dependent oxidation of precorrin‐2 to sirohydrochlorin, but also the insertion of iron into this oxidized intermediate, thereby producing siroheme. Thus CysG is a multifunctional protein solely responsible for siroheme synthesis from uro'gen III in E. coli, and accordingly is renamed siroheme synthase.
MAP kinase activation during heat shock in quiescent and exponentially growing mammalian cells
Authors: Dubois Marie-Françoise , Bensaude Olivier
Published: July 2002

In numerous cases of signal transduction, the mitogen‐activated protein kinases (MAP kinases) or extracellular regulated kinases (ERKs) are found to be activated by phosphorylations which result in electrophoretic mobility changes. Activities of MAP kinases in cytosolic extracts can also be monitored by the capacity of such extracts to phosphorylate myelin basic protein. These two assays were used to demonstrate that MAP kinases were rapidly activated during heat shock of both quiescent and exponentially growing mammalian (hamster, rat, mouse and human) cells. Thus, the MAP kinase cascade is likely to also ensure heat‐shock signal transduction and contribute to the regulation of the complex array of metabolic changes designated as the heat‐shock response.
Cell surface receptor directed targeting of toxin to human malaria parasite, Plasmodium falciparum
Authors: Surolia Namita , Misquith Sandra
Published: July 2002

Gelonin (a toxin and type II ribosome inactivating protein) when linked to human transferrin can be targeted to Plasmodium falciparum. The transferrin toxin conjugate is significantly toxic to parasite growth and is 25 times more potent than toxin alone in inhibiting parasite protein synthesis. The mechanism of its entry into the intraerythrocytic parasite is discussed.
Pregnancy and cervical infection with human papillomaviruses
Authors: Morrison E.A.B. , Gammon M.D. , Goldberg G.L. , Vermund S.H. , Burk R.D.
Published: July 2002

Objective: This investigation was undertaken to assess whether pregnancy represents a risk factor for human papillomavirus (HPV) infection which is independent of age and other known HPV risk factors. Methods: One hundred eighty‐nine women were enrolled from the outpatient clinics of a large municipal hospital. The subjects completed a self‐administered questionnaire and underwent a gynecologic examination which included a cervicovaginal lavage. Lavage samples were assessed for the presence of HPV DNA by restriction enzyme analysis and Southern blot hybridization. Statistical significance was assessed by the χ2‐test. Logistic regression was used to assess the association between pregnancy and HPV while controlling for the effect of other risk factors. Results: The prevalence odds ratio (POR) for the association of pregnancy and HPV infection was 2.2 (95% C.I. 1.1–4.5). The prevalence of HPV increased with increasing gestational age (HPV prevalence of 18.9% among non‐pregnant women vs. 27.3% in those in the first 12 weeks of pregnancy and 39.7% in those who were past the 12th week of pregnancy). Although statistical significance was not achieved in a multivariate model which controlled for age, race/ethnicity, education, age at first coitus, number of sexual partners within the last year and parity, the POR associated with the current pregnancy (POR = 2.1) was not substantially changed by correcting for these risk factors, suggesting that these factors were not significant confounders of the association between pregnancy and HPV infection. Conclusion: Current pregnancy is associated with a modestly increased prevalence of cervical HPV infection. This association appears to be independent of age and other major HPV risk factors.