bluuebunny/crossref_metadata_2025 huggingface dataset:
ADP‐ and epinephrine‐elicited release of [3H]guanylylimododiphosphate from platelet membranes
Authors: Motulsky Harvey J. , Insel Paul A.
Published: July 2002

Epinephrine‐promoted release of [3H]guanylylimidodiphosphate ([3H]Gpp(NH)p) from human platelet membranes has been used to probe the interactions between alpha2‐adrenergic recpetors and Ni, the guanine nucleotide binding protein that couples those receptors to an inhibition of adenylate cyclase activity. We show here that ADP, which also acts through specific platelet receptors to inhibit adenylate cyclase activity, also promotes the release of [3H]Gpp(NH). The amount of [3H]Gpp(NH)‐release elicited by epinephrine and by ADP together is equal to the sum of the amounts released by the two agents acting individually. Furthermore the maximal amounts of [3H]Gpp(NH)‐release elicited by each of the two agents approximates the numbers of receptors for ADP and epinephrine present in the platelet membranes. These results suggest that the two receptor types interact with distinct portions of the pool of Ni molecules and that each receptor initiates guanine‐nucleotide exchange on a single molecule of Ni.
The stimulation of macrophage prostaglandin E2 and thromboxane B2 secretion by Streptococcus mutans insoluble glucans
Authors: Abiko Yoshimitsu , Shibata Yasuko , Fukushima Kazuo , Murai Seidai , Takiguchi Hisashi
Published: July 2002

The effect of insoluble glucan synthesized by Streptococcus mutans on [3H]arachidonate metabolites secretion from peritoneal macrophages was studied. Insoluble glucans stimulated [3H]arachidonate release and secretion of prostaglandin E2 and thromboxane B2 from macrophages. In contrast, commercial soluble glucan (dextran) did not induce [3H]arachidonate release.
Possible involvement of NADPH‐cytochrome P450 reductase and cytochrome b5 on β‐ketostearoyl‐CoA reduction in microsomal fatty acid chain elongation supported by NADPH
Authors: Nagao Masaki , Ishibashi Teruo , Okayasu Takako , Imai Yoh
Published: July 2002

The chain elongation products from [2‐14C]malonyl‐CoA and palmitoyl‐CoA by rat liver microsomes initiated by NADPH were found mainly to be 18:1 and 18:0. When anti‐NADPH‐cytochrome P450 reductase or anti‐cytochrome b
5 immunoglobulin G were included in this system, not only was the overall chain elongation significantly suppressed, but also a new radioactive product, which was identified as 2‐heptadecanone derived from an intermediate β‐ketostearate, is accumulated depending upon the amounts of IgG added. These results suggest that NADPH‐cytochrome P450 reductase and cytochrome b
5 participate in the conversion from β‐ketostearoyl‐CoA to β‐hydroxystearoyl‐CoA, which is the first reductive step of the microsomal chain elongating system.
Na+‐dependent proline transport in isolated membrane vesicles from the L6 muscle cell line
Authors: Klip Amira , Gagalang Enriqueta , Logan William J.
Published: July 2002

Membrane vesicles of L6 myoblasts were prepared in order to study the amino acid transport system A. The role of the membrane in the adaptive response of transport to amino acid‐supplementation was assessed. The membranes, prepared by N2 cavitation, displayed Na+ (but not K+)‐dependent L‐proline uptake. An overshoot of L‐[3H]proline uptake was observed after exposure of the vesicles to an inward Na+ gradient. Isolated membrane vesicles loaded with 50 μM proline displayed countertransport (stimulation of proline uptake). It is concluded that the adaptive decrease of proline uptake observed in amino acid‐supplemented cells cannot be accounted for by trans‐inhibition of transport.
Role of histones H1 and H3 in the maintenance of chromatin in a compact conformation
Authors: Marion Christian , Roux Bernard , Coulet Pierre R.
Published: July 2002

Chromatin polynucleosomes have been digested with trypsin immobilized on collagen membranes. This method allows the mild removal of the most accessible histone fragments simply by dipping the enzymatic membrane into the chromatin solution, without modification of its ionic and chemical composition. These results demonstrate that the removal of H1 does not affect the higher‐order structure of chromatin and that only the elimination of the terminal regions of H3 leads to the unfolding of H1‐depleted fibres. This observation suggests that structural changes reported in many previous works were not due to only the removal of H1 but to a concomitant unbinding from DNA of the N‐terminal domain of H3.
Identification of antigenic components of Toxoplasma gondii by an immunoblotting technique
Authors: Partanen Paul , Turunen Hannu J. , Paasivuo Raili , Forsblom Erik , Suni Jukka , Leinikki Pauli O.
Published: July 2002

Proteins of Toxoplasma gondii were separated by SDS‐polyacrylamide gel electrophoresis with subsequent transfer to a nitrocellulose sheet by electrophoretic blotting. Immunologically reactive polypeptides were detected by human sera with previously known toxoplasma antibody levels. Heavy chain‐specific, peroxidase‐conjugated anti‐human immunoglobulins were used as the indicator antibodies for the separate identification of IgG and IgM reactive polypeptides. IgG toxoplasma antibodies reacted with several antigens of M
r ≈27 000–67 000, while toxoplasma‐specific IgM seemed to detect only a few polypeptides. The M
r of 35 000 for the dominating IgM reactive polypeptide was observed.
Wheat mitochondrial DNA encodes a eubacteria‐like initiator methionine transfer RNA
Authors: Gray Michael W. , Spencer David F.
Published: July 2002

An initiator methionine transfer RNA (tRNAf
Met) gene has been identified by sequence analysis of a cloned SalI restriction fragment of wheat mitochondrial DNA. The 3′‐end of this gene, which does not encode the CCA terminus of the mature tRNA, is separated by only one basepair from the 5′‐end of the 18 S ribosomal RNA gene, suggesting that the two genes are part of the same transcriptional unit. The wheat mitochondrial tRNAf
Met sequence, the first from plant mitochondria, displays strong structural affinity with eubacterial/chloroplast tRNAf
Met sequences.
Assembly of microtubules with ATP: evidence that only a fraction of the protein is assembly‐competent
Authors: Islam Khalid , Burns Roy G.
Published: July 2002

Chick brain microtubule protein can be assembled in vitro with ATP, although the extent of assembly is less than that with GTP. The ATP‐induced assembly is not the result of generation of GTP by the co‐purifying nucleoside diphosphate kinase. Neither an observed increase in the critical concentration nor the phosphorylation of MAP2 can account for the decreased extent of assembly. However, whereas microtubules are formed with both ATP and GTP, incubation with ATP yields additional filaments and polymorphic aggregates. The results demonstrate that of the total protein which can be assembled into microtubules by GTP, about 25–35% is assembled into other structural forms in the presence of ATP.
A spin‐labeled derivative of Procion brilliant blue MX‐R
Authors: Porumb Tudor , Lascu Ioan , Porumb Horea , Pop Radu D. , Bucsa Liliana
Published: July 2002

ESR
Nucleoside diphosphate kinase
Anthraquinone trimme dye
Differential spectroscopy
Phospholransferase active site
Platelet‐activating factor stimulates phospholipase in quiescent Swiss mouse 3T3 fibroblast
Authors: Kawaguchi Hideaki , Yasuda Hisakazu
Published: July 2002

Platelet‐activating factor(PAF) induced the rapid hydrolysis of phospholipids in Swiss mouse 3T3 fibroblasts prelabeled with [3H]arachidonic acid or [3H]choline. Up to 23% of the [3H]arachidonic acid incorporated in phospholipids was released into the medium when PAF was added to cells. We also observed increases of diacylglycerol and lysophosphatidylcholine in the medium and cells after the addition of PAF. These results support the possibility that platelet‐activating factor stimulates phospholipase A2 and C activities in 3T3 cells.
Assignment of the Fe‐Nge(His)stretching mode in the resonance Raman spectra of a monomeric insect cyanomethaemoglobin
Authors: Kerr Ellen A. , Yu Nai-Teng , Gersonde Klaus
Published: July 2002

Resonance Raman (RR) spectra of the monomeric cyanomethaemoglobin CTT III from insect larvae of Chironomus thummi thummi are shown for the range of 200–550 cm−1. By iron and cyanide isotope exchange a line varying between 307 cm−1 for 57Fe‐13C15N and 311 cm−1 for 54Fe‐12C14N, has been assigned to the Fe‐Nϵ stretching mode of this haem complex. The substitution of 54Fe for 57Fe has no effect on the Fe‐C = N bending mode whereas it affects the Fe‐CN stretching mode.
Rat Thy‐1 antigen has a hydrophobic segment at the carboxyl terminus
Authors: Moriuchi Tetsuya , Silver Jack
Published: July 2002

We have isolated a cDNA clone coding for rat Thy‐1 antigen from a rat thymocyte cDNA library. The 549 base pairs insert includes the complete coding region of a mature Thy‐1 polypeptide of 142 amino acids, 31 amino acids longer than the previously reported glycoprotein sequence. These 31 amino acids contain an extremely hydrophobic region of 26 uncharged amino acids which apparently represent the transmembrane segment that allows for integration within the membrane. The presence of this additional protein segment will solve most of the enigmas regarding the properties of the C‐terminal region of Thy‐1 antigen.
The effect of lipopolysaccharide on lipid bilayer permeability of β‐lactam antibiotics
Authors: Hiruma Ryoichi , Yamaguchi Akihito , Sawai Tetsuo
Published: July 2002

The lipid‐bilayer permeability of cephalosporins was extensively suppressed by addition of lipopolysaccharide to liposomal membrane in proportion to the hydrophobicity of the drugs. This suggests that the polysaccharide chain layer contributes to the barrier function. The importance of the polysaccharide chain in the barrier function was also supported by the fact that the permeability to Rd‐type lipopolysaccharide‐containing liposomes showed essentially the same dependency on the hydrophobicity of the cephalosporins as that of the lipopolysaccharide‐free liposomes. In this case the permeability of the cephalosporins was proportional to their hydrophobicity. Similar lipopolysaccharide effect was also observed in the permeation of penicillins.
Thrombin induces the rapid formation of inositol bisphosphate and inositol trisphosphate in human platelets
Authors: Siess Wolfgang , Binder Helge
Published: July 2002

Human platelets prelabeled with [3H]inositol were exposed to thrombin. The aqueous soluble inositol phosphates were separated by anion exchange column chromatography, paper chromatography or high‐performance liquid chromatography, and identified by cochromatography with authentic standard substances. Thrombin immediately induces the rapid formation of inositol 1,4‐bisphosphate and inositol 1,4,5‐trisphosphate. Accumulation of inositol‐1‐monophosphate and inositol‐2‐monophosphate occurs later after a time lag of 10 sec. The results indicate that the phospholipase C induced polyphosphoinositide hydrolysis rather than the phosphatidylinositol hydrolysis is the triggering event for platelet activation, and support the concept of inositol 1,4,5‐trisphosphate as putative second messenger.
Quantification of picogram levels of specific DNA immobilized in microtiter wells
Authors: Nagata Yoshiho , Yokota Hiroshi , Kosuda Osamu , Yokoo Keiko , Takemura Kazuo , Kikuchi Tetsuya
Published: July 2002

A non‐radioisotopic and sensitive method for quantification of specific DNA immobilized in microtiter wells has been developed. This method is based upon the immobilization of DNA in microtiter wells and hybridization with biotinylated DNA probe which is followed by complexing with avidin‐β‐galactosidase. By measuring fluorescence emitted from the hydrolyzed product by β‐galactosidase of 4‐methylumbelliferyl‐β‐D‐galactoside, it has become possible to quantify a few picograms of specific DNA in DNA samples immobilized in plastic microtiter wells.
The interaction between phenylalanine rings in proteins
Authors: Singh J. , Thornton J.M.
Published: July 2002

An analysis has been made of the geometry of phenylalanine‐phenylalanine interactions in proteins of known structure. 162 Phe‐Phe interactions were found with C‐C distances less than 4.6 Å. Three angles were used to define the geometry of interaction, P = the angle betwen ring planes, and polar coordinates, Tθ, Tϕ to specify the relative spatial disposition of the two rings. The overall distribution of P values is the same as that expected for a random distribution of planes in 3 dimensions; i.e. the majority, of interactions have P approaching 90°. However, for high Tθ values (when one Phe lies directly above the ring of the other Phe) the distribution is non‐random, and a preference for perpendicular interactions is expressed. This preference is in accord with recent quantum‐mechanical calculations.
Facilitated transport of 3‐O‐methyl‐D‐glucose in human polymorphonuclear leukocytes
Authors: Okuno Yasuhisa , Plesner Liselotte , Larsen Thomas R. , Gliemann Jørgen
Published: July 2002

Transport of the nonmetabolizable hexose analogue 3‐O‐methyl‐D‐glucose (3OMG) was measured in human polymorphonuclear leukocytes at 37° pH 7.4. 30MG at very low concentration (0.05 mM) equilibrated with the intracellular water with a rate constant of about 0.08 s−1. Transport of 3OMG in the presence of 20 μM cytochalasin B and transport of L‐glucose were insignificant. Countertransport of 14C‐labelled 3OMG was demonstrated. Exchange of 3OMG between the extracellular and intracellular water showed saturation with a K
m of about 4 mM. Thus, the transport of 3OMG is mediated almost exclusively by facilitated diffusion.
Gene conversion in human immunoglobulin γ locus shown by unusual location of IgG allotypes
Authors: Lefranc Marie-Paule , Helal Ahmed-Noureddine , de Lange Gerda , Chaabani Hassan , van Loghem Erna , Lefranc Gérard
Published: July 2002

The constant region of the γ1, γ2 and γ3 heavy chains of the human IgGl, IgG2 and IgG3 immunoglobulins carries antigenic determinants or G1m, G2m and G3m allotypes, which are genetic markers of these subclasses. The exceptional presence on γl and γ2 chains of Gm allotypes usually located on the CH3 domain of γ3 shows an unexpected clustering of base changes and subsequent identity of short DNA sequences in the CH3 exon of the non‐allelic γ1,γ2 and γ3 genes. Such clusters of substitutions are not easily explained on the classical basis of point mutations. A gene conversion, which substituted a segment of the γ1 or γ2 gene with the homologous region of the non‐allelic γ3 gene, is more likely. Other examples of possible conversion involving the γ genes are described. The conservation or the restoration of short sequences produced by the conversion events might be related to the biological properties of the constant region of the heavy chains.
Inhibition of fungal and mammalian sterol biosynthesis by 2‐aza‐2,3‐dihydrosqualene
Authors: Ryder Neil S. , Dupont Marie-Claude , Frank Ingeborg
Published: July 2002

2‐Aza‐2,3‐dihydrosqualene (I) and a quaternary ammonium derivative (II) inhibited ergosterol biosynthesis in cells and cell‐free extracts of the pathogenic yeast Candida albicans as measured by incorporation of radiolabelled precursors. The compounds inhibited squalene epoxidase and 2,3‐oxidosqualene cyclase to varying degrees in microsomes from C. albicans and from rat liver. The rat liver epoxidase was 50% inhibited by I at 2.4 μM. In C. albicans cells, but not in cell‐free extracts, I also inhibited lanosterol demethylation and led to accumulation of an unidentified polar product.
Activation of a D‐form of rabbit muscle glycogen synthase by Ca2+‐activated protease
Authors: Hiraga Akira , Tsuiki Shigeru
Published: July 2002

Glycogen synthase isolated from rabbit skeletal muscle as a D‐form (synthase D2) is activated by a rat liver cytosolic protein differing from any of the known protein phosphatases (D2 activase). Although reversible by phosphorylation by cyclic AMP‐dependent protein kinase, the activation is a result of limited proteolysis by D2 activase, which has been identified as the Ca2+‐activated protease.
Inhibition of protein synthesis by the β‐subunit of spectrin
Authors: Kudlicki Wieslaw , Kramer Gisela , Hardesty Boyd
Published: July 2002

The 220 kDa β‐subunit of erythroid cell spectrin is a potent inhibitor of protein synthesis in lysates from rabbit reticulocytes. On the basis of weight of protein added to a lysate reaction mixture, it has about half the inhibitory activity of highly purified heme‐regulated eIF‐2α kinase. Inhibition appears to be at the level of peptide initiation but does not involve a kinase that phosphorylates eIF‐2 on its α‐subunit.
Characterisation of a high‐affinity VIP receptor in human lung parenchyma
Authors: Schachter M. , Dickinson K.E.J. , Miles C.M. , Sever P.S.
Published: July 2002

A method is described for preparing human lung parenchymal membranes essentially free of carbon contamination. Using this technique, a high‐affinity 125I‐VIP‐binding site has been characterised. The receptor density is approx. 200 protein and the K
d of 125I‐VIP by saturation binding is 200 pM. The dissociation kinetics are complex and cannot be described by first‐order kinetics. Several VIP‐related peptides displace 125I‐VIP from this binding site with a rank order of potency: VIP > rat GRF > PHM> PHI > human GRF>secretin>glucagon. Displacement curves of these peptides exhibited slope factors significantly less than unity with the exception of human GRF.
A secretable serine proteinase with highly restricted specificity from cytolytic T lymphocytes inactivates retrovirus‐associated reverse transcriptase
Authors: Simon Hans-Georg , Fruth Ulli , Kramer Michael D. , Simon Markus M.
Published: July 2002

TSP‐1, a murine T cell specific proteinase, is expressed in cytolytic T lymphocytes and secreted upon their interaction with antigen bearing target cells. In searching for possible extracellular substrates of the enzyme in the physiological environment of cytolytic effector cells, we have investigated the proteolytic activity of TSP‐1 on retroviral proteins. It is shown that reverse transcriptase derived from the retrovirus Moloney murine leukemia virus is inactivated by TSP‐1 via limited proteolysis. The data suggest the possibility that cytolytic T lymphocytes are able to interfere with retroviral replication by secreting a serine proteinase which degrades viral proteins.
The structure of procalcitonin of the salmon as deduced from its cDNA sequence
Authors: Pöschl E. , Lindley I. , Hofer E. , Seifert J.-M. , Brunowsky W. , Besemer J.
Published: July 2002

Oligonucleotide probes based on the known amino acid sequence of salmon calcitonin were used to screen a cDNA library obtained from ultimobranchial glands of salmon for clones encoding salmon calcitonin. From the cDNA sequence of strongly hybridizing clones the complete primary structure of the calcitonin precursor could be deduced. Its overall structure is identical with the structures of procalcitonins from other vertebrates and has the highest homology with the chicken precursor.
Molecular architecture of secretin receptors: The specific covalent labelling of a 51 kDa peptide after cross‐linking of [125I]iodo‐secretin to intact rat pancreatic acini
Authors: Gossen Denis , Poloczek Piotr , Svoboda Michal , Christophe Jean
Published: July 2002

p‐Azidophenylglyoxal (APG), a heterobifunctional reagent with one group reacting selectively with arginine residues and another group photoactivable, was used to cross‐link [125I]secretin prebound to intact rat pancreatic acini. The best yield was obtained when the [125I]secretin‐acini complex was incubated under dim light with 2 mM APG at 37°C and pH 8.0, followed by photolysis at 312 nm. The main secretin binding peptide cross‐linked under reducing conditions, when tested by SDS‐PAGE and autoradiography: (i) had a molecular mass of 51 kDa and was not a subunit of a larger disulfide‐linked structure, and (ii) was distinct from the main VIP binding peptide coexisting in the same preparation.
A new heparin‐inhibited and polyamine‐activated protein kinase from bovine kidney
Authors: Singh Toolsee J.
Published: July 2002

Two casein kinases, casein kinase‐1 (CK‐1) and casein kinase‐2 (CK‐2), have been characterized from many sources. In this study we describe the properties of a third casein kinase, designated casein kinase‐3 (CK‐3). CK‐3 (M
r 32 000) is readily separated from CK‐2 by gel filtration and from CK‐1 by hydroxyapatite chromatography. CK‐3 phosphorylates several proteins, including phosphorylase kinase. Phosphorylation of phosphorylase kinase by CK‐3 results in a 10‐fold enzyme activation. CK‐3 is activated by spermine and inhibited by heparin, ADP, and divalent metal ions (Mn2+, Zn2+). Heparin inhibition of the kinase is reversed by spermine. The physical and regulatory properties of CK‐3 are very similar to CK‐1, suggesting that these kinases may be closely related.
Identification of the high‐molecular‐mass mitochondrial oxaloacetate keto‐enol tautomerase as inactive aconitase
Authors: Belikova Yu.O. , Kotlyar A.B. , Vinogradov A.D.
Published: July 2002

The homogeneous bovine heart mitochondrial high‐molecular‐mass oxaloacetate keto‐enol tautomerase [(1988) Biochim. Biophys. Acta 936, 10–19] is shown to be an iron‐sulfur protein as revealed by the enzyme spectral properties and direct chemical determination of non‐heme iron and acid‐labile sulfur. The protein is capable of catalysing the aconitase reaction after treatment with ferrous ions under anaerobic conditions. Treatment of the ‘activated’ protein with N‐ethylmaleimide results in the simultaneous irreversible loss of the oxaloacetate keto‐enol tautomerase and aconitase activities. The effects of some substrates and inhibitors on both activities show that the same catalytic site is involved in the oxaloacetate tautomerase and aconitase reactions. It is concluded that the protein previously described as a 80 kDa oxaloacetate keto‐enol tautomerase is inactive aconitase.
A phosphatase from chloroplast stroma of Nicotiana tabacum hydrolyses 2′‐carboxyarabinitol 1 ‐phosphate, the natural inhibitor of Rubisco to 2′‐carboxyarabinitol
Authors: Gutteridge Steven , Julien Bryan
Published: July 2002

An enzyme has been identified in the stroma of tobacco chloroplasts that degrades the natural inhibitor of Rubisco, 2′‐carboxyarabinitol 1‐phosphate (2CA1P). The products of the catalysis, determined by HPLC and phosphate analysis are 2′‐carboxyarabinitol and inorganic phosphate indicating that the enzyme is a phosphatase. The only other phosphate esters found to be degraded by the enzyme were the branched‐chain carboxylic sugar bisphosphates, 2′‐carboxyarabinitol and ribitol 1,5‐bisphosphates that were converted to the corresponding 5‐monophosphates. Other intermediary metabolites of the photosynthetic cycle were essentially unaffected by the phosphatase. The kinetic parameters of the enzyme are consistent with those required to relieve the inhibition of rubisco in vivo.
Monitoring protein crystallization by dynamic light scattering
Authors: Mikol V. , Hirsch E. , Giegé R.
Published: July 2002

Crystallization of lysozymes induced by temperature lowering has been monitored by dynamic light scattering from the onset of supersaturation to the growth of protein crystals up to a noticeable size (150–200 μm). The apparent size of the scatterers was found to increase up to a maximum value as supersaturation proceeded, then to decrease down to its initial value. Apparitions of crystals (20–30 μm) were observed during this decrease. Light scattering is thus proved to be a sensitive technique to follow protein crystallization and to provide useful information on the process.
Nucleotide sequence of the second psbG gene in Synechocystis 6803
Authors: Mayes Steve R. , Cook Katie M. , Barber James
Published: July 2002

Nucleotide sequencing of the second Synechocystis 6803 psbG gene, psbG2 shows the predicted polypeptide to be 219 amino acids long. It is less similar to chloroplast psbG genes than is the Synechocystis
psbG1 copy. Alignment with seven other psbG protein sequences, including that from the Paramecium mitochondrial genome reveals a central highly conserved region common to each. This is discussed as evidence supporting the proposal that the psbG polypeptide is a NAD(P)H dehydrogenase (complex I) subunit in cyanobacteria, chloroplasts and mitochondria.
Influence of the aminoacyl‐tRNA synthetase inhibitors and the diadenosine‐5'‐tetraphosphate phosphonate analogues on the catalysis of diadenosyl oligophosphates formation
Authors: Biryukov A.I. , Zhukov Yu.N. , Lavrik O.I. , Khomutov R.M.
Published: July 2002

Well‐known aminoacyl‐tRNA synthetase (ARSase) inhibitors, namely the analogues of amino acids and aminoacyl adenylates (aminoalkyl‐ and aminophosphonyl adenylates with K
i ⋍ 0.1 μM) as well as the diadenosine 5',5'''‐p
1,p
4‐tetraphosphate (Ap4A) phosphonoanalogues, were for the first time used for the Ap4A biosynthesis regulation. Effects of a set of such compounds on lysyl‐, phenylalanyl‐ and alanyl‐tRNA synthetases from E. coli, capable of synthesizing Ap4A in the presence of Zn2+ ions and pyrophosphatase, have been studied. The adenylate analogues were found to inhibit the Ap4A and Ap3A formation (I
50 ⋍ 6 mM). Aminophosphonic and aminophosphonous acids are not involved in Ap3A and Ap4A biosynthesis and inhibited it at high concentrations. The Ap4A phosphoanalogues slightly inhibited the major reactions of ARSases, as well as the biosynthesis of Ap3A and Ap4A, at a concentration of 5 mM.
Induction of nitric oxide synthase by cytokines in vascular smooth muscle cells
Authors: Busse Rudi , Mülsch Alexander
Published: July 2002

We investigated the mechanisms by which cytokines lead to a diminished responsiveness of vascular smooth muscle to vasoconstrictors. The attenuation of noradrenaline‐induced contraction by 6 to 24 h incubations with the cytokines, tumor necrosis factor and interleukin‐1, in endotheliumdenuded rabbi, aorta was associated with an increase in intracellular cyclic GMP level. This increase was abolished by the stereoselective inhibitor of nitric oxide‐synthase. N
o‐nitro‐L‐arginine and by cycloheximide. Formation of nitric oxide was detected in the cytosol of cytokine‐treated native and cultured smooth muscle cells by activation of purified soluble guanylate cyclase, and depended on tetrahydrobiopterin, but not on ? ‐calmodulin. The results indicate that cytokines induce a nitric oxide‐synthase of the macrophage‐type in vascular smooth muscle.
Partial purification and characterization of the human erythrocyte Mg2+ ‐ATPase A candidate aminophospholipid translocase
Authors: Morrot Gil , Zachowski Alain , Devaux Philippe F.
Published: July 2002

A Mg2+‐ATPase‐enriched fraction was obtained from solubilized human erythrocyte membranes by ammonium sulphate precipitation and anion‐exchange chromatography. The solubilized enzyme, of apparent molecular weight 120 kDa, requires phosphatidylserine to be fully active. Phosphatidylethanolamine but not other anionic phospholipids can only partially restore the activity. The Mg‐ATPase has a low affinity for Mg2+‐ATP and is inhibited by fluoride, vanadate, vanadyl and calcium ions. From these characteristics, we infer that this Mg2+‐ATPase is the same protein as the aminophospholipid translocase which regulates the membrane phospholipid transverse distribution in human erythrocytes by actively transporting aminophospholipids from the outer to the inner monolayer.
How do viral reverse transcriptases recognize their RNA genome?
Authors: Mesnard Jean-Michel , Lebeurier Geneviéve
Published: July 2002

Reverse transcription is not solely a retroviral mechanism. Hepadnaviruses and caulimoviruses have RNA intermediates that are reverse transcribed into DNA. Moreover non‐viral retroelements, retrotransposons, use reverse transcription in their transposition. All these retroelements encode reverse transcriptase but each group developed their own expression modes capable of assuring a specific and efficient replication of their genomes.
2D crystal forms of annexin IV on lipid monolayers
Authors: Newman R.H. , Leonard K. , Crumpton M.J.
Published: July 2002

Two‐dimensional crystalline arrays of annexin IV were generated by interaction of the purified protein with a phospholipid monolayer. Image analysis of electron micrographs of the protein crystals, which diffracted to 3.5 nm respectively, revealed p6 and p3 symmetry. Annexin IV gave two crystal forms with unit cells of 18 × 18 nm and 28 × 28 nm. The former unit cells wax similar to a previously described form of annexin VI. The implications of those observations are discussed.
Ca2+‐storage organelles
Authors: Krause Karl-Heinz
Published: July 2002

Intracellular Ca2+‐storage organelles are found in virtually all eukaryotic cells. They play an important role in the regulation of the cytosolic free Ca2+ concentration and, thereby, in the regulation of cellular activity. Ca2+‐storage organelles consist, in the simplest model of a Ca2+ pump, of a Ca2+‐storage protein and a Ca2+‐release channel. The primary structure of these functionally important proteins of Ca2+‐storage organelles is similar in different cell types and conserved through evolution. In contrast, their spatial arrangement and, thus, the architecture of Ca2+‐storage organelles may vary dramatically from one cell type to another.
Up‐regulation of the interleukin‐6‐signal transducing protein (gp130) by interleukin‐6 and dexamethasone in HepG2 cells
Authors: Schooltink Heidi , de Leur Hildegard Schmitz-Van , Heinrich Peter C. , Rose-John Stefan
Published: July 2002

The hepatic IL‐6‐receptor is composed of an 80 kDa IL‐6‐binding protein and a 130 kDa polypeptide (gp130) believed to be involved in signal transduction. Previous experiments have shown that the 80 kDa IL‐6‐rueptor is up‐regulated by glucocorticoids, but not by IL‐6. Here we demonstrate that IL‐6 together with the synthetic glucocorticoid dexamethasone induces the expression of mRNA for gp130 approximately 5‐fold in HepG2 cells. The induction was dose‐ and time‐dependent, Dexamothasone alone, interferon‐γ, IL‐1α and IL‐1β had no effect. A possible role for the regulation of the IL‐6‐signal transducing protein gp130 in various inflammatory states is proposed.
Separation of gate‐ and channel‐forming domains in the pore‐forming protein of the outer membrane of Pseudomonas aeruginosa
Authors: Yoshihara Eisaku , Nakae Taiji
Published: July 2002

The domains of the pore‐forming protein responsible for the gate and channel formations were separated and identified in the outer membrane of Pseudomonas aeruginosa. The proteolytic cleavage of the 46K channel protein, protein D2, yielded two major domains with apparent M
r of 27K and 19K. We identified the 27K polypeptide to be the channel‐forming domain by an in vitro permeability assay. The channel size of purified 27K domain was indistinguishable from that of native protein D2. Degradation of the 19K domain into small subfragments increases the channel activity about ten times suggesting that the 19K polypeptide forms the gate or cap.
The choline transporter is the major site of control of choline oxidation in isolated rat liver mitochondria
Authors: Kaplan Claude P. , Porter Richard K. , Brand Martin D.
Published: July 2002

The degree of control exerted by the mitochondrial choline transporter over the choline oxidation pathway was measured in isolated rat liver mitochondria. Choline transporter activity was titrated with hemicholinium‐3, a known competitive inhibitor of the transporter. It was shown that the rate of betaine efflux from mitochondria was an accurate measure of choline oxidation. The relative rate of choline oxidation was measured as a function of the relative degree of inhibition of the transporter. The resulting data gave a flux control coefficient over choline oxidation of 0.9 for the choline transporter. It is concluded that the choline transporter is the major site for control of choline oxidation in isolated rat liver mitochondria.
Cloning and expression of novel isoforms of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase from bovine heart
Authors: Vidal Hubert , Crepin Karine M. , Rider Mark H. , Hue Louis , Rousseau Guy G.
Published: July 2002

Distinct 6‐phosphofructo‐2‐kinase (PFK‐2)/fructose 2,6‐bisphosphatase (FBPase‐2) cDNAs were cloned from bovine heart, showing that PFK‐2/FBPase‐2 gene B, which contains 16 exons, codes for at least five mRNAs. Three of them (B1, B2, B4) could encode the 58,000‐M
r isozyme. In B2 mRNA, exon 15 encodes four more residues than in Bl. In B4 mRNA, exon 15 encodes six more residues than in B1, butexon 16 (20 residues) is missing. B3 mRNA corresponds to the 54,000‐M
r isozyme. It lacks exon 15 and also differs from the other mRNAs in the 5' noncoding region. B5 mRNA encodes a truncated form. When expressed in E. coli, the recombinant isoforms corresponding to all these mRNAs except B5 exhibited PFK‐2 activity.
Storage of saposins A and D in infantile neuronal ceroid‐lipofuscinosis
Authors: Tyynelä Jaana , Palmer David N. , Baumann Marc , Haltia Matti
Published: July 2002

We have isolated storage cytosomes from brain tissue of patients with infantile neuronal ceroid‐lipofuscinosis. The purified storage bodies were subjected to compositional analysis which revealed a high content of proteins, accounting for 43% of dry weight. Saposins A and D, also known as sphingolipid activator proteins (SAPs), were shown to constitute a major portion of the accumulated protein using gel electrophoresis and sequence analysis. This is the first time that saposins have been found to be stored in any form of neuronal ceroid‐lipofuscinosis.
Participation of tyrosine kinase in capping, internalization, and antigen presentation through membrane immunoglobulin in BAL17 B lymphoma cells
Authors: Shimo Kuniaki , Gyotoku Yuichi , Arimitsu Yoshiko , Kakiuchi Terutaka , Mizuguchi Junichiro
Published: July 2002

BAL17 cells pulsed with goat anti‐IgM or anti‐IgD as antigens stimulated a goat IgG specific T cell clone in terms of inositol phosphate production. The antigen‐presenting capacity of BAL17 cells was inhibited by pretreatment with the tyrosine kinase inhibitors herbimycin A or genistein. Furthermore, ligand‐induced capping and endocytosis of membrane immunoglobulin, monitored at the single cell level, was also blocked by herbimycin A. These results indicate that tyrosine phosphorylation plays an important role in receptor‐mediated antigen presentation by B cells.
ATP‐sensitive K+ channels in heart muscle cells first open and subsequently close at maintained anoxia
Authors: Thierfelder Stephan , Doepner Bettina , Gebhardt Christine , Hirche Hansjürgen , Benndorf Klaus
Published: July 2002

In ventricular myocardial cells of the guinea pig and the mouse, anoxia caused after a mean latency of 439 ± 141 s and 129 ± 23 s (mean ± S.E.M.), respectively, a large current through KATP‐channels. This current disappeared within several seconds when reoxygenating the cells but decayed also completely at maintained anoxia. The kinetics of the latter process, however, were much slower and obeyed an approximately monoexponential time course with time constants in the range of 30 s. The results suggest that in the ischaemic myocardium KATP‐channels contribute only to the initial phase of extracellular K+ accumulation.
The sevenmaker gain‐of‐function mutation in p42 MAP kinase leads to enhanced signalling and reduced sensitivity to dual specificity phosphatase action
Authors: Bott Cynthia M. , Thorneycroft Simon G. , Marshall Christopher J.
Published: July 2002

A mammalian mutant MAP kinase, D319N ERK2, analogous to Drosophila melanogaster sevenmaker (rlsem) gain‐of‐function mutation was shown to have an increased sensitivity to low levels of signalling in vivo. However, the mutation does not lead to an elevated basal kinase activity and still requires activation by MAP kinase kinase (MAPKK) as does wild type ERK2. This increased responsiveness seen in vivo is not due to an increased ability to phosphorylate substrates but appears to reflect a reduced sensitivity to a MAP kinase phosphatase CL100.
Primary structure of a multimeric protein, homologous to the PEP‐utilizing enzyme family and isolated from a hyperthermophilic archaebacterium
Authors: Cicicopol Christiana , Peters Jürgen , Kellermann Josef , Baumeister Wolfgang
Published: July 2002

A large protein complex (approx. 2000 kDa) was found in the cytosol of the hyperthermophilic archaebacterium Staphylothermus marinus. The purified protein was shown to be a homomultimer of 93 kDa subunits, the primary structure of which was determined by nucleotide sequence analysis. The protein belongs to the family of phosphoenolpyruvate‐utilizing enzymes and represents the first member characterized in archaebacteria. Its homomultimeric organisation differs from the typically dimeric structure of its eubacterial and eukaryotic counterparts.
Isolation of a cDNA clone specifying rat chaperonin 10, a stress‐inducible mitochondrial matrix protein synthesised without a cleavable presequence
Authors: Ryan Michael T. , Hoogenraad Nicholas J. , Høj Peter B.
Published: July 2002

We have isolated a cDNA clone encoding chaperonin 10 from rat liver. The cDNA specifies a protein of 102 amino acids which, when transcribed and translated in vitro, yields a single basic product (pI > 9) that co‐migrates exactly with the heat shock inducible cpn10 of rat hepatoma cells during 2D gel‐electrophoresis. It is concluded that cpn10, unlike the majority of nuclear‐encoded proteins of the mitochondrial matrix, is synthesised without a cleavable targeting signal and that, following removal of the initiating methionine, it becomes acetylated prior to mitochondrial import. Incubation of 3H‐ or 35S‐labelled cpn10 with mitochondria confirms these conclusions and shows that cpn10 is imported into mitochondria in an energy‐dependent process which is inhibited by the presence of 2,4‐dinitrophenol.
Identification of reactive lysines in phosphoenolpyruvate carboxykinases from Escherichia coli and Saccharomyces cerevisiae
Authors: Bazaes Sergio , Goldie Hughes , Cardemil Emilio , Jabalquinto Ana María
Published: July 2002

Escherichia coli and Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases (PEPCKs), were inactivated by pyridoxal 5′‐phosphate followed by reduction with sodium borohydride. Concomitantly with the inactivation, one pyridoxyl group was incorporated in each enzyme monomer. The modification and loss of activity was prevented in the presence of ADP plus Mn2+. After digestion of the modified protein with trypsin plus protease V‐8, the labeled peptides were isolated by reverse‐phase high‐performance liquid chromatography and sequenced by gas‐phase automatic Edman degradation. Lys286 of bacterial PEPCK and Lys289 of the yeast enzyme were identified as the reactive amino acid residues. The modified lysine residues are conserved in all ATP‐dependent phosphoenolpyruvate carboxykinases described so far.
Stimulatory effect of unsaturated fatty acids on the level of plasminogen activator inhibitor‐1 mRNA in cultured human endothelial cells
Authors: Karikó Katalin , Rosenbaum Helga , Kuo Alice , Zurier Robert B. , Barnathan Elliot S.
Published: July 2002

To determine whether unsaturated fatty acids induce changes in the mRNA level of plasminogen activator inhibitor type‐1 (PAI‐1), Northern analyses were performed on human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells that were treated with two common fatty acids. Supplementation of cultured HUVEC with docosahexanoic acid (DHA) or with dihomogamma linolenic acid (DGLA), resulted in a concentration dependent, specific increase of the PAI‐1 transcript levels, which was detectable within 2 h. DHA and DGLA treatment of smooth muscle cells did not result in changes in the PAI‐1 mRNA levels. Homology search of the upstream regulatory region of the PAI‐1 gene sequences identified a consensus nucleotide sequence for a fatty acid‐responsive element. Our results indicate that unsaturated fatty acids selectively increase PAI‐1 mRNA levels in endothelial cells, the primary source of circulating PAI‐1 in vivo.
Clustered localization of oligomeric Nef protein of human immunodeficiency virus type 1 on the cell surface
Authors: Fujii Yoichi , Otake Kaori , Fujita Yoshikazu , Yamamoto Naoki , Nagai Yoshiyuki , Tashiro Masato , Adachi Akio
Published: July 2002

We studied human immunodeficiency virus type 1 (HIV‐1) Nef protein biochemically and histologically. HIV‐1 Nef, derived from baculosystem and from cells infected with HIV‐1, formed homomeric monomers, dimers, trimers, and further polymers. These oligomers were non‐covalently associated. In cells infected with HIV‐1, Nef molecules were clustered at the cell surface as well as cytoplasm. Our previous results have indicated that the Nef on the surface of cells infected with HIV‐1 is cytotoxic against uninfected CD4+ T cells. Thus, it is very likely that the HIV‐1‐mediated cytotoxic reaction is due, at least in part, to the clustered localization of oligomeric Nef on the cell surface.
Oral contraceptives and venous thromboembolism
Authors: Speroff L.
Published: July 2002

In October, 1995, the United Kingdom Committee on Safety of Medicines recommended that women should use oral contraceptives containing desogestrel or gestodene only if prepared to accept an increased risk of thromboembolism. This action was based on observational studies that indicated a 2–3‐fold increase in the risk of thromboembolism when desogestrel and gestodene‐containing contraceptives were compared to products with levonorgestrel. The fact that these studies point in the same direction is concerning, but it is possible that these observational studies, somewhat similar in design, are influenced by the same unrecognized biases. Furthermore, the case numbers are relatively small (20–40 cases), and, for example, preferential prescribing to women at greater risk could affect the results. It is difficult to reconcile the conclusions with the strong belief supported by good evidence that thrombosis is an estrogen dose‐related complication, and that progestational agents have no impact on clotting parameters. In my view, these equivocal reports are not of sufficient strength to escape biases and to change our prescribing of oral contraceptives.
Effect of tumor necrosis factor α and β on human oligodendrocytes and neurons in culture
Authors: McLaurin J. , D'Souza S. , Stewart J. , Blain M. , Beaudet A. , Nalbantoglu J. , Antel J.P.
Published: July 2002

Cytokines produced by infiltrating hematogenous cells or by glial cells activated during the course of central nervous system disease or trauma are implicated as mediators of tissue injury. In this study, we have assessed the extent and mechanism of injury of human‐derived CNS oligodendrocytes and neurons in vitro mediated by the cytokines tumor necrosis factor α and β and compared these with the tumor necrosis factor independent effects mediated by activated CD4+ T‐cells. We found that activated CD4+ T‐cells, but not tumor necrosis factor α or β, could induce significant release of lactate dehydrogenase, a measure of cell membrane lysis, from oligodendrocytes within 24 hr. Neither induced DNA fragmentation as measured using a fluorescence nick‐end labelling technique. After a more prolonged time period (96 hr), tumor necrosis factor α did induce nuclear fragmentation changes in a significant proportion of oligodendrocytes without increased lactate dehydrogenase release. The extent of DNA fragmentation was comparable to that induced by serum deprivation. Tumor necrosis factor β effects were even more pronounced. In contrast to oligodendrocytes, the extent of DNA fragmentation, assessed by propidium iodide staining, induced in neurons by tumor necrosis factor α was less than that induced by serum deprivation. In‐situ hybridization studies of human adult glial cells in culture indicated that astrocytes, as well as microglia, can express tumor necrosis factor α mRNA.
Rapid neurite formation in a human cortical neuronal cell line
Authors: Dunn K.J. , Perez‐Polo J.R. , Wood T.G.
Published: July 2002

The subclone HCN‐1 was derived from parental cell lines from cortical tissue of a patient with unilateral megalencephaly growth and immunochemistry staining characteristics [G. V. Ronnett et al. (1990) Human cortical neuronal cell line: establishment from a patient with unilateral megalencephaly. Science248, 603–605]. As we and others have shown, HCN‐1A cells can be induced to differentiate to a neuronal‐like morphology. HCN‐1A cells stain positively for neurofilament, neuron‐specific enolase and the low‐affinity neurotrophin receptors, p75NGFR, but not for myelin basic protein, S‐100, or glial fibrillary acidic protein (GFAP). HCN‐1A cells also stain positively for gamma‐aminobutyric acid and glutamate. In the present study, we examine the effects of cell density on the requirements for efficient induction of differentiation of HCN‐1A cells and analyze the time course of this induction and its reversion. We also characterize the changes in cytoskeletal proteins of HCN‐1A cells in response to their differentiation neuronal phenotype.
Human fetal brain cells in aggregate culture: A model system to study regulatory processes of the developing human neuropeptide Y (NPY)‐producing neuron
Authors: Aguila‐Mansilla Nelson , Barnea Ayalla
Published: July 2002

Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the brain and it has been implicated in a wide range of brain functions, including mentation. The aim of this study was to establish a culture system of human fetal brain cells expressing NPY in a regulated manner. The NPY production in response to forskolin and phorbol 12‐myristate 13‐acetate (PMA) was taken as a criterion for regulated expression of NPY. Aggregates were formed from dissociated cells derived from the cerebral hemispheres of human fetuses (12.5–19 weeks' gestation) by constant rotation and were maintained in serum‐free medium. A 24 hr exposure to 10 μM forskolin + 20 nM PMA led to a 2–6‐fold increase in NPY content of the cultures, most of which (80–90%) was secreted into the medium. The latter consisted of two substances differing in size: one corresponding in size to proNPY and the other to NPY. Thus, forskolin + PMA led to an increased production of NPY. Exposure to PMA alone led to an increase in NPY production comparable to that seen after forskolin + PMA and this effect of PMA was dose‐dependent. In contrast, forskolin alone did not induce NPY production. Conditioned medium, derived from monolayer cultures enriched with human astrocytes, enhanced NPY production in response to forskolin + PMA in an age‐dependent manner. The NPY production by aggregates derived from a 12.5‐week‐, 14‐week‐ and 18‐week‐old fetus was enhanced 3‐3.6‐fold, 1.6‐2‐fold and 1.1‐fold, respectively. Thus, expression of the NPY neurons in this culture system is a regulated process. The NPY production is enhanced markedly by activation of the protein kinase C pathway and by an astrocyte‐derived soluble substance(s). Based on these results, we propose that this culture system can serve as a model for the study of regulatory processes of the human developing NPY neuron.