bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of erogenous quinones with photosystem II in inside‐out thylakoids
Authors: Delrieu Marie-José , Rosengard Françoise
Published: July 2002

The presence of erogenous quinones PPBQ, DCBQ and DMQ in inside‐out thylakoids alters the oscillation pattern with period 4 of the flash‐induced fluorescence yield. The results can be interpreted by assuming that at ambient temperature PPBQ replaces PQ in 100% of the PS II centers detected by fluorescence, DMQ and DCBQ being bound to 40–60% of these centers only. The evaluation of the percentage of double hits in a 3 μs flash allows estimation of the electron‐transfer rate from Q‐
A to Fe3+, which varies from 9 μs (PPBQ) to 20μs (DMQ).
Differential activation by fMet‐Leu‐Phe and phorbol ester of a plasma membrane phosphatidylcholine‐specific phospholipase D in human neutrophil
Authors: Gelas Pascale , Ribbes Gérard , Record Michel , Terce François , Chap Hugues
Published: July 2002

Signal transduction involving phosphatidylcholine hydrolysis has been investigated in human neutrophils (PMN) after in situ generation of [3H]alkylacyl‐sn‐glycero‐3‐phosphocholine [3H]alkylacyl‐GPC) by cell incubation with [3H]alkylacetyl‐GPC. When PMN were stimulated with the chemotactic peptide N‐formyl‐Met‐Leu‐Phe (fMLP) or phorbol myristate acetate (PMA) in the presence of cytochalasin B, both 1‐O‐alkyl‐2‐acyl‐sn‐glycero‐3‐phosphate (PA) and 1‐O‐alkyl‐2‐acyl‐sn‐glycerol (AAG) were generated. On addition of the agonists in the presence of ethanol, phosphatidylethanol (PE) was formed with a concomitant decrease in PA and AAG. These results indicate the presence of a phospholipase D (PLD) acting on phosphatidylcholine in human PMN. The kinetics of hydrolysis were quite different according to the stimulus. Whereas fMLP induced a maximum rise in PA and AAG at 30–45 s, these products began to appear only after 1 min upon cell incubation with PMA. Similar amounts of products were formed at 1 min with fMLP and only at 5 min with PMA. Although similar time courses of PA generation were obtained in the absence of cytochalasin B, AAG were no longer involved and therefore cannot account for intracellular second messenger under physiological conditions. Subcellular distribution studies demonstrated the exclusive location of PA and PE in the plasma membrane. The possible involvement of PA in respiratory burst activation is discussed.
Direct activation of GTP‐binding regulatory proteins (G‐proteins) by substance P and compound 48/80
Authors: Mousli M. , Bronner C. , Landry Y. , Bockaert J. , Rouot B.
Published: July 2002

The neuropeptide substance P and the polyamine compound , both known to activate mast cell secretory processes, increased the rate of GTPγS binding to G‐proteins purified from calf brain (Go/Gi mixture). The GTPase activity of G‐proteins was also increased by substance P and compound in a dose‐dependent and Mg2+‐dependent way. These effects were similar to those of the wasp venom peptide rnastoparan, another histamine releaser of rat peritoneal and human skin mast cells. This suggests that the secretory property of compound and substance P is not due to a receptor‐mediated process but, like mastoparan, results from a direct activation of G‐proteins.
Component of the alternative oxidase localized to the matrix surface of the inner membrane of plant mitochondria
Authors: Rasmusson Allan G. , Møller Ian M. , Palmer John M.
Published: July 2002

In mitoplasts from Arum maculatum spadices, succinate dehydrogenase (EC 1.3.99.1) and the alternative, cyanide‐resistant oxidase activity (measured as m‐chlorobenzhydroxamic acid‐sensitive duroquinol oxidation) was unaffected by treatment with trypsin. In contrast, when 85% inside‐out submitochondrial particles were treated with trypsin the alternative oxidase activity was inhibited by about 50% and succinate dehydrogenase activity by about 40%. Thus, a trypsin‐sensitive component of the alternative pathway is located on the inner surface of the inner mitochondrial membrane. After trypsin treatment of the inside‐out submitochondrial particles the inhibited alternative oxidase activity was partly restored by including 0.7 M citrate in the assay medium. This indicates that trypsin does not destroy the active site but merely causes a conformational change in the enzyme, thereby lowering its activity.
Structure‐activity relationship in vasoconstrictor effects of sarafotoxins andendothelin‐1
Authors: Kitazumi Kazuhiro , Shiba Toshiharu , Nishiki Katsuyuki , Furukawa Yasuo , Takasaki Chikahisa , Tasaka Kenji
Published: July 2002

Sarafotoxins (SRTa, SRTb and SRTc) as well as endothelin‐1 (ET‐1) produced vasoconstrictions in rat thoracic aorta, rat isolated perfused mesentery and pithed rat in various of extents. The potency was ET‐1 > SRTb > SRTa > SRTc at lower doses, but SRTb > ET‐1 > SRTa > SRTc at higher doses. [Nitrophenylsulfenylated Trp21]SRTb and SRTb(1‐19) caused no vasoconstriction. Either the reduction and carboxymethylation of Cys residues, the destruction of the intramolecular loop or the production of the non‐natural disulfide bond, eliminated the constrictor activity. These results indicate that Trp21 and the intramolecular loop structure are essential, and Lys9 and Tyr13 may play some important roles for the vasoconstrictor activity of these peptides.
Electron microscopic demonstration of a common structural motif in human complement factor C3 and rat α1 ‐inhibitor 3 (murinoglobulin)
Authors: Ikai Atsushi , Nishigai Masaaki , Saito Akio , Sinohara Hyogo , Muto Yutaka , Arata Yoji
Published: July 2002

Electron microscopy of two homologous giant proteins revealed that complement factor C3 and αi‐inhibitor 3 have a common structural motif of a semicircularly bent string 18–20 nm long with two or three bumps indicating globular domains. C3 had a structure similar to the letter C with a small but distinct hole in the center. α1‐Inhibitor 3 was a more complete ring sometimes ajar at one corner. When the latter was treated with a proteinase, it became slightly flattened and adopted a squarish C‐shape.
Gap junctional permeability is affected by cell volume changes and modulates volume regulation
Authors: Ngezahayo Anaclet , Kolb Hans-Albert
Published: July 2002

Isolated pancreatic acinar cell pairs became electrically uncoupled by exposure to a mild hypotonic shock. Reduction of bath osmolarity caused a delayed closure of gap junctional channels in the minute range. Dialysis of cell pairs by GTP[S] in the double whole‐cell patch‐clamp mode shortened the latency and shifted the hypotonically induced electrical uncoupling to lower osmolarity changes. Cellular treatment with cytochalasin B catalyzed electrical uncoupling by a hypotonic shock. In all cases, electrical uncoupling could be blocked completely by the protein kinase C (PKC) inhibitor polymyxin B. These results provide the first evidence suggesting that changes of cell volume and gap junctional permeability are correlated and that a G‐protein dependent mechanism is involved. Evidence is presented that gap juntional coupling modulates volume regulation.
A reversible carrier mediates the transport of malate at the tonoplast of Catharanthus roseus cells
Authors: Bouyssou H. , Canut H. , Marigo G.
Published: July 2002

The release was prevented by the anion transport inhibitor DIDS indicating the reversibility of the carrier.
Activation of protein kinase C by phorbol esters induces DNA synthesis and protein phosphorylations in glomerular mesangial cells
Authors: Issandou Marc , Darbon Jean-Marie
Published: July 2002

The tumor‐promoting phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) is shown to be mitogenic for quiescent glomerular mesangial cells cultured in serum‐free conditions. TPA induces DNA synthesis measured by PH thymidine incorporation in a dose‐dependent manner with an ED10 of 7 ng/ml and an optimal response for 50 ng/ml. The phorbol ester action is potentiated by insulin with an increase of the maximal effect from 232 ± 15% of TPA alone to 393 ± 96% for TPA plus insulin. Down‐regulation of protein kinase C by prolonged exposure to TPA completely abolishes the mitogenic effect of the phorbol ester. Using a highly resolutive 2D electrophoresis, we have shown that TPA is able to stimulate the phosphorylation of 2 major proteins of M
1 80000, pI 4.5 (termed 80K) and M
1 28000, pI 5.7–5.9 (termed 28K). The 80K protein phosphorylation is time‐ and dose‐dependent with an ED10 of 8 ng/ml TPA. Exposure or mesangial cells to heat‐shock induces synthesis of a 28K protein among a set of other proteins suggesting that the 28K protein kinase C substrate belongs to the family of low molecular mass stress proteins. Mitogenic concentrations of TPA and phorbol 12,13‐dibutyrate inhibit [125I]epidermal growth factor binding and stimulate the 80K protein phosphorylation with the same order of potency. The inactive tumor‐promoter 4x‐phorbol was found to be ineffective both on these 2 parameters and on DNA synthesis. These results suggest a positive role of protein kinase C on mesangial cell proliferation and indicate the existence in this cell line of 2 major protein kinase C substrates.
Identification of seven novel protein‐tyrosine kinase genes of Drosophila by the polymerase chain reaction
Authors: Shishido Emiko , Emori Yasufumi , Saigo Kaoru
Published: July 2002

We used the polymerase chain reaction to identify 7 novel tyrosine‐kinase genes (dtk1 to −7) in Drosophila melanogaster dtk4 coded for a part of the kinase catalytic domain nearly identical in sequence to that of the human receptor for insulin‐like growth factor 1, whereas sequences encoded by dtk1 and dtk2 were highly homologous to that of the chicken fibroblast growth factor receptor.
Overexpression of alcohol oxidase in Pichia pastoris
Authors: de Hoop M.J. , Cregg J. , Keizer-Gunnink I. , Sjollema Klaas , Veenhuis M. , Ab G.
Published: July 2002

The protein import capacity of peroxisomes in methylotrophic yeasts was studied using Pichia pastoris containing one or two extra copies of the gene encoding the peroxisomal protein alcohol oxidase. The alcohol oxidase overproduced in this strain was only partially imported and assembled into the active, octameric form of the protein. The excess remained in the cytosol as protein aggregates composed of monomers. These results are discussed in view of the possible application of peroxisomes as storage compartments for heterologous proteins.
A gene family homologous to the S‐phase specific gene in higher plants is essential for cell proliferation in Saccharomyces cerevisiae
Authors: Ito Masaki , Yasui Akira , Komamine Atsushi
Published: July 2002

Previously we reported the isolation and characterization of the gene, cyc07, which was specifically expressed in the S phase during the cell cycle in synchronous cell division cultures of the higher plant, Catharanthus roseus. We found that the yeast Saccharomyces cerevisiae contains two closely related genes which show a high degree of similarity (about 64% at the amino acid level) to cyc07 of C. roseus. Site‐directed disruption mutations demonstrated that the two yeast genes, homologous to cyc07, constitute an essential gene family for cell proliferation in yeast cells. Furthermore, the rate of cell proliferation varied with the gene copy number.
Biotinyl analogues of amylin as biologically active probes for amylin/CGRP receptor recognition
Authors: Chantry Andrew , Foot Elizabeth A. , Leighton Brendan , Day Anthony J. , Willis Antony C.
Published: July 2002

Biotinyl analogues of rat amylin were synthesised with sulfosuccinimidyl 2‐(biotinamido)ethyl‐1,3‐dithiopropionate(NHS‐SS‐Biotin). Biotinylated amylin peptides were purified by HPLC, quantitated, and the presence of the biotin group at Lys‐1 confirmed by peroxidase‐labelled avidin and FAB mass spectroscopy. Amylin‐biotin retained a similar affinity for binding to rat liver plasma membranes compared with rat amylin and also completely inhibited insulin‐stimulated glycogen synthesis in rat soleus muscle incubated in vitro. These biologically active amylin probes will enable a complete analysis of amylin/CGRP receptor expression in various cell types and facilitate the isolation and characterisation of the hormone‐receptor complex.
Resonance effects in strongly exothermic long‐range electron transfer and their possible implications for the behaviour of site‐directed mutant proteins
Authors: Christensen Hans E.M. , Conrad Lars S. , Hammerstad-Pedersen Jan M. , Ulstrup Jens
Published: July 2002

Long‐range electron transfer investigations of hemoproteins, blue copper and iron‐sulphur proteins frequently rest on electronically excited metall centres. When the excitation energy approaches the oxidation or reduction potentials of intermediate residues the superexchange view normally used, however, fails and a variety of new dynamic features arise. These all involve population of the intermediate cation or anion residue states which can be partially or wholly vibrationally relaxed. We discuss suitable views and a new theoretical formalism for these phenomena. We also note some important implication for site‐directed mutagenesis in long‐range, strongly exothermic electron transfer processes.
Characterization of carbohydrates in the α2‐macroglobulin receptor
Authors: Jensen Poul H. , Gliemann Jørgen , Ørntoft Torben
Published: July 2002

Carbohydrates were characterized in the human placental α2‐macroglobulin receptor and its associated protein. Carbohydrates, largely N‐linked, contributed to about 18% of the size of the receptor α‐chain and to about 25% of the β‐chain. The 40 kDa receptor‐associated Protein also contained carbohydrate. The α‐ and β‐chains contained a wide variety of carbohydrates as judged by binding of lectins. Monosaceharide‐competing inhibition of α2M‐methylamine binding by WGA suggested a functional significance of sugars in binding of ligands to the α‐chain.
Carbon‐by‐carbon discrimination of 13C incorporation into liver fatty acids
Authors: Cunnane S.C.
Published: July 2002

Quantitative carbon‐by‐carbon analysis would be useful in determining the origin and fate of carbons involved in fatty acid metabolism. Incorporation of 13C from 2‐[13C]acetate into specific carbons of liver fatty acids was lowest at the n—2 carbon of saturates and monoenes but was 47% greater at acyl Cl than at C2, suggesting substantial redistribution of the 13C from C2 to Cl of acetyl CoA or malonyl CoA prior to 13C incorporation into fatty acids during de novo synthesis or during elongation. Thus, 13C derived from exogenous acetate can be quantitatively measured and is differentially incorporated into individual carbons depending on position in the fatty acid molecule.
Accumulation of protoporphyrin IX in light‐sensitive mutants of Escherichia coli
Authors: Miyamoto Kazumasa , Nishimura Koichi , Masuda Tatsuru , Tsuji Hideo , Inokuchi Hachiro
Published: July 2002

The accumulation of protoporphyrin IX (Proto IX) in light‐sensitive mutants of Escherichia coli was detected by spectrofluorimetry. Fluorescence emission and excitation spectra were recorded from extracts of bacterial cells. Proto IX clearly accumulated in cells with mutations in the visA (hemH) gene but not in the wild‐type strain CA274 or in visA mutants that had been rendered light‐resistant by introduction of the wild‐type visA
+ gene. Accumulation of Proto IX was also not observed in cells with a mutation in the visB gene. These results confirm the hypothesis that the sensitivity of the visA mutants to light is caused by the abnormal accumulation of Proto IX, a substrate of ferrochelatase, as the result of a genetic defect in the gene for ferrochelatase.
Stereospecific assignments of the leucine methyl resonances in the 1H NMR spectrum of Lactobacillus casei dihydrofolate reductase
Authors: Ostler G. , Soteriou A. , Moody C.M. , Khan J.A. , Birdsall B. , Carr M.D. , Young D.W. , Feeney J.
Published: July 2002

A general method is described for the stereospecific assignment of methyl resonances in protein NMR spectra based on selective deuteration procedures. A selectively deuterated dihydrofolate reductase from L. casei was prepared by incorporating stereoselectively deuterated l‐leucine, (2S,4R)[5,5,5‐2H3]leucine. By comparing the COSY spectra of the dihydrofolate reductase‐methotrexate complexes formed using deuterated and non‐deuterated enzyme the stereospecific assignments for resonances of all 13 leucine residues were obtained by noting the absence of cross‐peaks in spectra from the deuterated proteins.
α2‐Macroglobulin bait region integrity
Authors: Gettins Peter G.W. , Beechem Joseph M. , Crews Brenda C.
Published: July 2002

To determine whether integrity of the bait region affects the structure of the remainder of human α2‐macroglobulin (α2M), we have determined the separation between cysteine residues in a methylamine‐reacted fast‐form of α2 M. From reduction in fluorescence intensity of covalently‐bound donor fluorophore caused by proximity to an acceptor, a separation of 35 ± 8 Å was calculated, which is identical to a previously determined value for proteinase‐treated fast‐form α2M. This indicates that although bait region cleavage is the physiological route to conformational change in α2M, bait region integrity per se does not significantly affect the structure of fast‐form α2M.
Phosphoryl exchange is involved in the mechanism of the insulin receptor kinase
Authors: Al-Hasani Hadi , Paßlack Waltraud , Klein Helmut W.
Published: July 2002

The cytoplasmic kinase domain of the human insulin receptor (IRKD; M
r 49 kDa) has been over‐expressed in insect cells using the baculovirus expression system. To investigate the kinase mechanism, we have compared the stoichiometry of ADP formation and phosphoryl transfer. After an initial phase of autophosphorylation, ATP is consumed without a stoichiometric increase in incorporated phosphate. During substrate phosphorylation using poly(Glu: Tyr) (4:1) phosphoryl transfer comes close to ATP turnover, which is independent of the presence of the substrate, indicating an increased efficiency (i.e. ATP turnover/phosphate incorporation) of phosphoryl transfer. Autophosphorylation under pulse‐chase conditions suggests the existence of a phosphoenzyme intermediate.
Stable expression of VLA‐4 and increased maturation of the β1‐integrin precursor after transfection of CHO cells with α4m cDNA
Authors: Jaspers Martine , de Meirsman Catherine , Schollen Els , Vekemans Sylvie , Cassiman Jean-Jacques
Published: July 2002

A full‐length cDNA coding for the murine α4 integrin subunit (α4m) was transfected into CHO‐K1 cells and cell lines that expressed VLA‐4 at their surface as a result of the association of transfected α4m with endogenous hamster β1 were selected. Functionality of the expressed α4mβ1 was shown by adhesion assays on VCAM‐1 and antibody (anti‐VCAM‐ 1) inhibition. Pulse chase experiments indicated that transfection of the murine α4 cDNA into CHO cells led to an increase in maturation and a decrease in degradation of the β1 precursor subunit compared to control CHO‐K1 cells. This was supported by FACS analysis, using an anti‐hamster β1 monoclonal antibody, which showed that more β1 subunit was expressed at the surface of these stably transfected α4m expressing cells. These results support the hypothesis that degradation of precursor β1 is at least partly determined by the quantity of α subunits available intracellulary for heterodimer formation.
Mutational analysis of Glu771 of the Ca2+‐ATPase of sarcoplasmic reticulum Effect of positive charge on dephosphorylation
Authors: Andersen Jens Peter
Published: July 2002

The glutamic acid residue Glu771 in the fifth transmembrane segment M5 of the Ca2+‐ATPase of rabbit fast twitch muscle sarcoplasmic reticulum was substituted with lysine, alanine, and glycine by site‐directed mutagenesis. Mutant Glu771→Lys was unable to occlude Ca2+, and Ca2+ did not inhibit phosphorylation from Pi or activate phosphorylation from ATP of this mutant. Mutants Glu771→Ala and Glu771→Gly were likewise unable to occlude Ca2+, but Ca2+ in the millimolar concentration range activated phosphorylation from ATP and inhibited phosphorylation from Pi of these mutants. The dephosphorylation of the ADP‐insensitive E2P phosphoenzyme intermediate of mutants Glu771→Ala and Glu771→Gly was found to be blocked, whereas the dephosphorylation proceeded rapidly for mutant Glu771→Lys. This finding suggests a role of the positive charge of the lysine in induction of dephosphorylation, supporting the hypothesis that the side chain of Glu771 participates in the countertransport of two protons per Ca2+‐ATPase cycle.
Cytochrome oxidase evolved by tinkering with denitrification enzymes
Authors: Saraste Matti , Castresana Jose
Published: July 2002

The cytochrome bc complex which is encoded by the fixNOPQ operon in Bradyrhizobiumjaponicum, is the most distant member of the haem‐copper cytochrome oxidase family. We have found that its major subunit, FixN, is homologous to the NorB subunit of nitric oxide reductase in a purple bacterium. A second evolutionary link between cytochrome oxidases and denitrification enzymes is the presence of a similar binuclear copper site in cytochrome aa
3 (the mitochondrial oxidase) and nitrous oxide reductase. This centre was probably acquired by a primitive FixN‐type oxidase, leading to the evolution of the mitochondrial‐type oxidase. These links suggest that the oxygen‐reducing respiratory chain developed from the anaerobic, denitrifying respiratory system.
Molecular mechanisms in synaptic vesicle recycling
Authors: De Camilli Pietro
Published: July 2002

Synaptic vesicles are specialized secretory organelles which are involved in the fast, point‐to‐point signaling typical of synapses. They store and secrete non‐peptide neurotransmitters and are continuously regenerated in nerve terminals by exoendocytotic recycling. This recycling represents a highly specialized form of the recycling pathway which occurs at the surface of all cells. Several unique properties make synaptic vesicles a powerful experimental model for studies of vesicular traffic. These unique properties include their abundance in brain, the high specialization of nerve terminals for synaptic vesicle recycling, the possibility of studying their exocytosis at the level of single events by electrophysiology and the availability of toxins which block their recycling. This lecture will summarize current information of molecular mechanisms in synaptic vesicle recycling with emphasis on recent studies carried out in my laboratory on mechanisms of vesicle reformation after exocytosis.
Translational fusion of heat labile enterotoxin chain B and β‐subunit of human chorionic gonadotropin: periplasmic expression in Escherichia coli and its immunogenicity
Authors: Pillai Devika , Dixit Aparna , Alok Deoraj , Garg Lalit C.
Published: July 2002

A fusion gene was constructed consisting of heat labile enterotoxin chain B (LTB) of E. coli genetically linked at its C‐terminus to the β‐subunit of human chorionic gonadotropin in translational fusion, under the control of tac promoter and LTB signal sequence. Expression of the fusion gene (about 5 μg/ml) in E. coli was confirmed by immunoblot analysis using both anti‐LTB and anti‐βhCG polyclonal antibodies. The fusion protein was efficiently processed and exported to the periplasmic space. LTB in the fusion protein retained its ability to bind to GM1 ganglioside receptor. Mice immunized with the fusion protein produce antibodies that recognize recombinant βhCG and the native hCG suggesting its potential use as a contraceptive vaccine.
Cytokine network in the central nervous system and its roles in growth and differentiation of glial and neuronal cells
Authors: Sawada Makoto , Suzumura Akio , Marunouchi Tohru
Published: July 2002

Cells resident within the central nervous system (CNS) can synthesize, secrete and respond to inflammatory cytokines not only contributing to the responses to injury or immunological challenge within the CNS, but also regulating their own growth and differentiation potential. The actions and cell communication via cytokines in the CNS are designated as the CNS cytokine network, in which microglia and astrocytes play the central roles. To further characterize the CNS cytokine network we investigated the differences in roles of these cells, and found that microglia might contribute to the early phase of cytokine production reaction and that astrocytes might contribute the late phase of the reaction. We also investigated roles of inhibitory cytokines such as TGF β, IL‐4, and IL‐10, and showed that each might play a distinct role in the inhibitory regulation in the CNS. We summarized our previous report about cellular distribution of cytokine receptors in the CNS cells and discussed their roles in the CNS cytokine network. Finally, we investigated that expression of IL‐6 and IL‐2 receptors in neuronal and oligodendrocytic differentiation, respectively. From these results, we discussed the features of the CNS cytokine network.
Involvement of CDSP 32, a drought‐induced thioredoxin, in the response to oxidative stress in potato plants
Authors: Broin Mélanie , Cuiné Stéphan , Peltier Gilles , Rey Pascal
Published: July 2002

In animal cells, yeast and bacteria, thioredoxins are known to participate in the response to oxidative stress. We recently identified a novel type of plant thioredoxin named CDSP 32 for chloroplastic drought‐induced stress protein of 32 kDa. In the present work, we measured comparable increases in the glutathione oxidation ratio and in the level of chlorophyll thermoluminescence, a specific marker for thylakoid lipid peroxidation in Solanum tuberosum plants subjected to drought or oxidative treatments (photooxidative stress, gamma irradiation and methyl viologen spraying). Further, substantial accumulations of CDSP 32 mRNA and protein were revealed upon oxidative treatments. These data show for the first time in plants the induction of a thioredoxin by oxidative stress. We conclude that CDSP 32 may preserve chloroplastic structures against oxidative injury upon drought.
Roles of p38 MAPK, PKC and PI3‐K in the signaling pathways of NADPH oxidase activation and phagocytosis in bovine polymorphonuclear leukocytes
Authors: Yamamori Tohru , Inanami Osamu , Nagahata Hajime , Cui Yu-Dong , Kuwabara Mikinori
Published: July 2002

Stimulation of bovine polymorphonuclear leukocytes (PMN) with serum‐opsonized zymosan (sOZ) induced the activation of p38 mitogen‐activated protein kinase (MAPK), protein kinase C (PKC) and phosphatidylinositol 3‐kinase (PI3‐K) and sOZ‐induced O2
− production was significantly attenuated by their inhibitors (SB203580 for p38 MAPK, GF109203X for PKC and wortmannin for PI3‐K). They caused significant attenuation of sOZ‐induced phosphorylation of p47phox as well. Flow cytometric analysis, however, revealed that SB203580 and wortmannin attenuated phagocytosis, but GF109203X facilitated it. The results suggest that p38 MAPK and PI3‐K participated in both signaling pathways of NADPH oxidase activation (O2
− production) and phagocytosis, and PKC participated in the signaling pathway of NADPH oxidase activation alone.
Requirement of sphingolipid α‐hydroxylation for fungicidal action of syringomycin E
Authors: Hama Hiroko , Young Debra A. , Radding Jeffrey A. , Ma Doreen , Tang Julia , Stock Stephen D. , Takemoto Jon Y.
Published: July 2002

Syringomycin E is an antifungal cyclic lipodepsinonapeptide produced by Pseudomonas syringae pv. syringae. To understand the mechanism of action of syringomycin E, a novel resistant Saccharomyces cerevisiae strain, BW7, was isolated and characterized. Lipid analyses revealed that BW7 contained only the hydrophobic subspecies of sphingolipids that are normally minor components in wild type strains. This aberrant sphingolipid composition was the result of lack of α‐hydroxylation of the amide‐linked very long chain fatty acids, suggesting a defective sphingolipid α‐hydroxylase encoded by the FAH1 gene. A yeast strain that lacks the FAH1 gene was resistant to syringomycin E, and failed to complement BW7. These results demonstrate that BW7 carries a mutation in the FAH1 gene, and that the lack of α‐hydroxylated very long chain fatty acids in yeast sphingolipids confers resistance to syringomycin E.
High CO2‐mediated down‐regulation of photosynthetic gene transcripts is caused by accelerated leaf senescence rather than sugar accumulation
Authors: Ludewig Frank , Sonnewald Uwe
Published: July 2002

The influence of elevated atmospheric CO2on transcript levels of photosynthetic genes was investigated in leaves ofNicotiana tabacumcv. SamsunNN and cv. Wisconsin38 plants. Plants were grown under ambient (400 ppm) and elevated (800/1000 ppm) atmospheric CO2, and transcript levels were determined in leaves of different age. Down‐regulation of photosynthetic gene transcripts was apparent in senescing leaves only. A correlation between transcript levels and leaf contents of soluble sugars could not be found. To investigate whether a shift in leaf ontogeny would be involved in the regulation of photosynthetic genes transgenic tobacco plants expressing either thegusoriptgene under control of the senescence‐specific SAG‐12 promoter [Gan, S. and Amasino, R.M. (1995) Science 270, 1986–1988] were included in our studies. As expected SAG‐12‐driven GUS activity increased with leaf age. This increase of GUS activity was stimulated by elevated atmospheric CO2, accompanied by a loss of chlorophyll and the down‐regulation of photosynthetic genes, verifying that high CO2accelerates leaf ontogeny. Senescence as well as down‐regulation of photosynthetic genes could be delayed byiptexpression. Levels of soluble sugars were indistinguishable from wild type or even slightly elevated inipttransgenic plants. Therefore, sugar accumulation as a cause for down‐regulation of photosynthetic genes under high CO2can be excluded. It appears more likely that the high CO2‐mediated decline in photosynthetic gene transcripts is due to a temporal shift in leaf ontogeny.
MSK1 is required for CREB phosphorylation in response to mitogens in mouse embryonic stem cells
Authors: Arthur J.Simon C , Cohen Philip
Published: July 2002

Mouse embryonic stem (ES) cells homozygous for disruption of the MSK1 gene had no detectable MSK1 activity. However, their activators (extracellular signal related kinase (ERK)1/ERK2) were stimulated normally in mitogen‐ and stress‐activated protein kinase (MSK)1−/− and wild type cells in response to tetradecanoylphorbol acetate (TPA) and epidermal growth factor (EGF). TPA and EGF induced the phosphorylation of cyclic AMP‐responsive element binding protein (CREB) at Ser‐133 and ATF1 at Ser‐63 in wild type cells and this was abolished by inhibition of the mitogen‐activated protein kinase cascade. In contrast, the TPA‐ and EGF‐induced phosphorylation of CREB/ATF1 was barely detectable in MSK1−/− cells. However, basal and forskolin‐induced phosphorylation was similar, indicating that the MSK1 ‘knockout’ did not prevent CREB phosphorylation by cyclic AMP‐dependent protein kinase. Thus MSK1 is required for CREB and ATF1 phosphorylation after mitogenic stimulation of ES cells.
Lymph node localisation of biodegradable nanospheres surface modified with poloxamer and poloxamine block co‐polymers
Authors: Hawley Ann E , Illum Lisbeth , Davis Stanley S
Published: July 2002

Studies were performed to develop a sub‐100 nm biodegradable colloidal system for the efficient delivery of drugs and diagnostic agents to the lymphatic system. Nanospheres of poly(lactide‐co‐glycolide) were prepared by interfacial polymer deposition. The nanospheres were coated with block co‐polymers in order to modify their surface characteristics. Radiolabelling of the nanospheres for in vivo tracing was achieved by the incorporation of the lipophilic complex 111In‐oxine during nanosphere preparation. In vitro stability of the radiolabelled nanospheres was determined in rat serum at 37°C. The lymphatic distribution of the nanospheres was determined after subcutaneous administration to the rat. Lymphatic uptake of all coated systems was enhanced compared to the uncoated nanospheres, and a maximal uptake of 17% of the administered dose in the regional lymph nodes was achieved. These observations suggest that the nanospheres are suitable for diagnostic and therapeutic applications in clinical and experimental medicine.
Tissue distribution and levels of gelsolin mRNA in normal individuals and patients with gelsolin‐related amyloidosis
Authors: Paunio Tiina , Kangas Hannele , Kiuru Sari , Palo Jorma , Peltonen Leena , Syvänen Ann-Christine
Published: July 2002

We measured quantitatively the mRNA levels of intracellular and secretory forms of gelsolin, an actin‐modulating protein, in human tissues from subjects of different ages. The intracellular gelsolin mRNA constituted the major type of gelsolin steady‐state mRNA in all tissues analyzed. Both forms of gelsolin were expressed in most adult tissues, with particularly high mRNA levels in all types of muscle and interestingly in skin. Between the adult and infantile tissues the most striking difference in expression levels was found in liver, as the adult liver contained only a subtle amount of gelsolin mRNA. Skin and muscle samples from patients with gelsolin‐related amyloidosis (FAF), with significantly increased concentrations of serum gelsolin, did not reveal an increased expression of the gene, and both mutant and wild‐type alleles were expressed in equal amounts. The high level of expression of the gelsolin gene in the skin in general could locally contribute to the characteristic skin amyloidosis in FAF patients.
Nitric oxide donors activate the cyclo‐oxygenase and peroxidase activities of prostaglandin H synthase
Authors: Maccarrone Mauro , Putti Sabrina , Finazzi Agrò Alessandro
Published: July 2002

Prostaglandin H synthase (PHS) is a dual enzyme with cyclo‐oxygenase and peroxidase activities. The nitric oxide (NO) donors, sodium nitroprusside (SNP), S‐nitroso‐N‐acetylpenicillamine (SNAP) and spermine NONOate (SPER/NO), activated both cyclo‐oxygenase and peroxidase activities of PHS. SNP activated PHS by increasing V
max without affecting K
m, the activation constants being 1.0 mM for cyclo‐oxygenase and 1.3 mM for peroxidase. Analysis of progress curves and absorption spectra of PHS suggested that NO released from SNP interacted with the heme at the active site of the enzyme. Moreover, SNP counteracted the peroxide‐induced inactivation of PHS, suggesting that the interplay between the intracellular peroxide and NO is critical in tuning PHS activity in cells.
Cyclophilin active site mutants have native prolyl isomerase activity with a protein substrate
Authors: Scholz Christian , Schindler Thomas , Dolinski Kara , Heitman Joseph , Schmid Franz X.
Published: July 2002

The prolyl isomerase activity of cyclophilins is traditionally measured by an assay in which prolyl cis/trans isomerization in a chromogenic tetrapeptide is coupled with its isomer‐specific cleavage by chymotrypsin. Two variants of mitochondrial cyclophilin with substitutions in the presumed active site (R73A and H144Q) are inactive in the protease‐coupled assay, but show almost wild‐type activity in an assay that is based on the catalysis of a proline‐limited protein folding reaction. This prolyl isomerase assay is preferable, both because coupling with proteolysis is avoided and because an intact protein instead of a short peptide is used as a substrate. Possibly, some earlier conclusions about the catalytic mechanism and the involvement of the prolyl isomerase activity in the cellular function of immunophilins may need reevaluation.
Oxidation of a critical methionine modulates DNA binding of the Drosophila melanogaster high mobility group protein, HMG‐D
Authors: Dow Linda K , Changela Anita , Hefner Heidi E , Churchill Mair E.A
Published: July 2002

HMG‐D is a major high mobility group chromosomal protein present during early embryogenesis in Drosophila melanogaster. During overexpression and purification of HMG‐D from E. coli, a key DNA binding residue, methionine 13, undergoes oxidation to methionine sulfoxide. Oxidation of this critical residue decreases the affinity of HMG‐D for DNA by three‐fold, altering the structure of the HMG‐D‐DNA complex without affecting the structure of the free protein. This work shows that minor modification of DNA intercalating residues may be used to fine tune the DNA binding affinity of HMG domain proteins.
Molecular cloning and characterization of two novel transport proteins from rat kidney
Authors: Schömig Edgar , Spitzenberger Folker , Engelhardt Martin , Martel Fátima , Örding Nicola , Gründemann Dirk
Published: July 2002

The recent cloning of renal transport systems for organic anions and cations (OAT1, OCT1, and OCT2) opened the possibility to search, via polymerase chain reaction (PCR) homology screening, for novel transport proteins. Two integral membrane proteins, UST1 and UST2, were cloned from rat kidney. RT‐PCR revealed that UST1 is confined to the kidney whereas UST2 mRNA was detected in all tested tissues. Sequence analyses suggest that UST1 and UST2, together with four related transporters, comprise, within the major facilitator superfamily, a so far unrecognized transporter family, termed amphiphilic solute facilitator (ASF) family. Characteristic signatures for the ASF family were identified.
The role of the C‐terminus for catalysis of the large thioredoxin reductase from Plasmodium falciparum
Authors: Gilberger Tim-Wolf , Bergmann Bärbel , Walter Rolf D , Müller Sylke
Published: July 2002

The thioredoxin system is one of the major thiol reducing systems of the cell. Recent studies have revealed that Plasmodium falciparum and human thioredoxin reductase represent a novel class of enzymes, which are substantially different from the isofunctional prokaryotic Escherichia coli enzyme. We identified the cysteines Cys88 and Cys93 as the redox active disulfide and His509 as the active site base [Gilberger, T.‐W., Walter, R.D. and Müller, S., J. Biol. Chem. 272 (1997) 29584–29589]. In addition to the active site thiols Cys88 and Cys93 the P. falciparum enzyme has another pair of cysteines at the C‐terminus: Cys535 and Cys540. To assess the possible role of these peripheral cysteines in the catalytic process the single mutants PfTrxRC535A and PfTrxRC540A, the double mutant PfTrxRC535AC540A and the deletion mutant PfTrxRΔ9 (C‐terminal deletion of the last nine amino acids) were constructed. All mutants are defective in their thioredoxin reduction activity, although they still show reactivity with 5,5′‐dithiobis (2‐nitrobenzoate). These data imply that the C‐terminal cysteines are crucially involved in substrate coordination and/or electron transfer during reduction of the peptide substrate.
Pore accessibility during C‐type inactivation in Shaker K+ channels
Authors: Basso Claudia , Labarca Pedro , Stefani Enrico , Alvarez Osvaldo , Latorre Ramon
Published: July 2002

Shaker K+ channels inactivate through two distinct molecular mechanisms: N‐type, which involves the N‐terminal domain and C‐type that appears to involve structural modifications at the external mouth of the channel. We have tested pore accessibility of the Shaker K+ channel during C‐type inactivation using Ba2+ as a probe. We determined that external Ba2+ binds to C‐type inactivated channels forming an extremely stable complex; i.e. there is Ba2+ trapping by C‐type inactivated channels. The structural changes Shaker channels undergo during C‐type inactivation create high energy barriers that hinder Ba2+ exit to either the extracellular solution or to the intracellular solution.
Specific activation of adenylyl cyclase V by a purinergic agonist
Authors: Pucéat Michel , Bony Claire , Jaconi Marisa , Vassort Guy
Published: July 2002

The present study was designed to investigate whether and how the purinergic stimulation of rat ventricular myocytes modulates the cAMP‐dependent pathway. Stimulation of cardiomyocytes with ATPγS in the presence of the phosphodiesterase inhibitor IBMX increases by 3‐fold intracellular cAMP content. In contrast to β‐adrenergic stimulation, the purinergic stimulation of adenylyl cyclase was not inhibited by activation or enhanced by inhibition of a Gi protein. Forskolin did not potentiate the effect of extracellular ATPγS on intracellular cAMP content but the effect of isoproterenol did. Like isoproterenol, the purinergic agonist decreased subsequent ADP‐ribosylation of a 45 kDa Gαs by cholera toxin. ATPγS also increased cAMP content in neonatal rat cardiomyocytes, a cell type that expresses a long form of Gs protein (αs, 52 kDa) in contrast to adult rat cardiomyocytes that express mostly a short form of Gs protein (αs, 45 kDa). Both purinergic and β‐adrenergic agonists increased cAMP in HEK 293 cells expressing type V adenylyl cyclase while cAMP was only increased by β‐adrenergic stimulation of HEK expressing type IV or VI adenylyl cyclases. Thus, we propose that the purinergic and β‐adrenergic stimulations differentially activate adenylyl cyclase isoforms in rat cardiomyocytes and that adenylyl cyclase V is the specific target of the purinergic stimulation.
The isolated proteolytic domain of Escherichia coli ATP‐dependent protease Lon exhibits the peptidase activity
Authors: Rasulova Fatima S , Dergousova Natalia I , Starkova Natalie N , Melnikov Edward E , Rumsh Lev D , Ginodman Lev M , Rotanova Tatyana V
Published: July 2002

Selective protein degradation is an energy‐dependent process performed by high‐molecular‐weight proteases. The activity of proteolytic components of these enzymes is coupled to the ATPase activity of their regulatory subunits or domains. Here, we obtained the proteolytic domain of Escherichia coli protease Lon by cloning the corresponding fragment of the lon gene in pGEX‐KG, expression of the hybrid protein, and isolation of the proteolytic domain after hydrolysis of the hybrid protein with thrombin. The isolated proteolytic domain exhibited almost no activity toward protein substrates (casein) but hydrolyzed peptide substrates (melittin), thereby confirming the importance of the ATPase component for protein hydrolysis. Protease Lon and its proteolytic domain differed in the efficiency and specificity of melittin hydrolysis.
Apocynin increases glutathione synthesis and activates AP‐1 in alveolar epithelial cells
Authors: Lapperre Thérèse S , Jimenez Luis A , Antonicelli Frank , Drost Ellen M , Hiemstra Pieter S , Stolk Jan , MacNee William , Rahman Irfan
Published: July 2002

Apocynin (4‐hydroxy‐3‐methoxy‐acetophenone) is a potent intracellular inhibitor of superoxide anion production in neutrophils. In this study, we studied the effect of apocynin on the regulation of the antioxidant glutathione (GSH) and activation of the transcription factor AP‐1 in human alveolar epithelial cells (A549). Apocynin enhanced intracellular GSH by increasing γ‐glutamylcysteine synthetase activity in A549 cells. Apocynin also increased the expression of γ‐GCS heavy subunit mRNA. This was associated with increased AP‐1 DNA binding as measured by the electrophoretic mobility shift assay. These data indicate that apocynin displays antioxidant properties, in part, by increasing glutathione synthesis through activation of AP‐1.
Binding partners for the myelin‐associated glycoprotein of N2A neuroblastoma cells
Authors: Strenge Karen , Schauer Roland , Kelm Sørge
Published: July 2002

The myelin‐associated glycoprotein (MAG) has been proposed to be important for the integrity of myelinated axons. For a better understanding of the interactions involved in the binding of MAG to neuronal axons, we performed this study to identify the binding partners for MAG on neuronal cells. Experiments with glycosylation inhibitors revealed that sialylated N‐glycans of glycoproteins represent the major binding sites for MAG on the neuroblastoma cell line N2A. From extracts of [3H]glucosamine‐labelled N2A cells several glycoproteins with molecular weights between 20 and 230 kDa were affinity‐precipitated using immobilised MAG. The interactions of these proteins with MAG were sialic acid‐dependent and specific for MAG.
αB‐crystallin in the rat lens is phosphorylated at an early post‐natal age
Authors: Ito Hidenori , Iida Kayo , Kamei Keiko , Iwamoto Ikuko , Inaguma Yutaka , Kato Kanefusa
Published: July 2002

We determined the developmental changes in the phosphorylation state of αB‐crystallin in lenses from rats at various post‐natal ages by isoelectric focusing gel electrophoresis or sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and a subsequent Western blot analysis of extracts of lenses using antibodies that recognized the carboxy‐terminal sequence or each of the three phosphorylated serine residues (Ser‐19, Ser‐45 and Ser‐59) in αB‐crystallin. Phosphorylated forms of αB‐crystallin were barely detected at birth but they became detectable at 3 weeks of age and reached plateau levels at 8 weeks of age. The phosphorylation of αB‐crystallin at Ser‐45 was observed preferentially. The active form of p44/42 MAP kinase, which is responsible for the phosphorylation of Ser‐45 in αB‐crystallin, also increased in a development‐dependent manner. Thus we found that the developmental increase of the phosphorylation at Ser‐45 of αB‐crystallin in the rat lens was due to the developmental activation of p44/42 MAP kinase.
Purification of Saccharomyces cerevisiae RNase H(70) and identification of the corresponding gene
Authors: Frank Peter , Braunshofer-Reiter Christa , Karwan Anneliese , Grimm Rudolf , Wintersberger Ulrike
Published: July 2002

We purified Saccharomyces cerevisiae RNase H(70) to homogeneity, using an optimized chromatographic purification procedure. Renaturation gel assay assigned RNase H activity to a 70 kDa polypeptide. Sequencing of tryptic peptides identified the open reading frame YGR276c on chromosome VII of the S. cerevisiae genome as the corresponding gene, which encodes a putative polypeptide of molecular mass of 62 849. We therefore renamed this gene RNH70. Immunofluorescence microscopy using a RNH70‐EGFP fusion construct indicates nuclear localization of RNase H(70). Deletion of RNH70 from the yeast genome did not result in any serious phenotype under the conditions tested. Homology searches revealed striking similarity with a number of eukaryotic proteins and open reading frames, among them the chimpanzee GOR protein, a homolog of a human autoimmune antigen, found to elicit autoimmune response in patients infected with hepatitis C virus.
Surface‐modified mutants of cytochrome P450cam: enzymatic properties and electrochemistry
Authors: Lo Kenneth Kam-Wing , Wong Luet-Lok , Hill H.Allen O
Published: July 2002

We report the electrochemistry of genetic variants of the haem monooxygenase cytochrome P450cam. A surface cysteine‐free mutant (abbreviated as SCF) was prepared in which the five surface cysteine residues Cys‐58, Cys‐85, Cys‐136, Cys‐148 and Cys‐334 were changed to alanines. Four single surface cysteine mutants with an additional mutation, R72C, R112C, K344C or R364C, were also prepared. The haem spin‐state equilibria, NADH turnover rates and camphor‐hydroxylation properties, as well as the electrochemistry of these mutants are reported. The coupling of a redox‐active label, N‐ferrocenylmaleimide, to the single surface cysteine mutant SCF‐K344C, and the electrochemistry of this modified mutant are also described.