bluuebunny/crossref_metadata_2025 huggingface dataset:
Botulinum ADP‐ribosyltransferase C3 but not botulinum neurotoxins C1 and D ADP‐ribosylates low molecular mass GTP‐binding proteins
Authors: Rösener S. , Chhatwal G.S. , Aktories K.
Published: July 2002

Botulinum ADP‐ribosyltransferase C3 modified 21‐24 kDa proteins in a guanine nucleotide‐dependent manner similar to that described for botulinum neurotoxin C1 and D. Whereas GTP and GTPγS stimulated C3‐catalyzed ADP‐ribosylation in the absence of Mg2+, in the presence of added Mg2+ ADP‐ribosylation was impaired by GTPγS. C3 was about 1000‐fold more potent than botulinum C1 neurotoxin in ADP‐ribosylation of the 21–24 kDa protein(s) in human platelet membranes. Antibodies raised against C3 blocked ADP‐ribosylation of the 21–24 kDa protein by C3 and neurotoxin C1 but neither cross reacted with neurotoxin C1 immunoblots nor neutralized the toxicity of neurotoxin C1 in mice. The data indicate that the ADP‐ribosylation of low molecular mass GTP‐binding proteins in various eukaryotic cells is not caused by botulinum neurotoxins but is due to the action of botulinum ADP‐ribosyltransferase C3. The weak enzymatic activities described for botulinum neurotoxins appear to be due to the contamination of C1 and D preparations with ADP‐ribosyltransferase C3.
Role of glycosylation in secretion of yeast acid phosphatase
Authors: Mrša Vladimir , Barbarić Slobodan , Ries Blanka , Mildner Pavao
Published: July 2002

The minimal glycosylation requirement for acid phosphatase secretion and activity was investigated using tunicamycin, an inhibitor of protein glycosylation, and a yeast mutant defective in the synthesis of oligosaccharide outer chains. The results obtained show that outer chain addition is not essential for secretion of active enzyme and that only 4 core chains, out of 8 normally attached to a protein subunit, are sufficient for enzyme transport to the periplasmic space. Enzyme forms with less than 4 chains were retained in membranes of endoplasmic reticulum. Secreted underglycosylated enzyme forms are partially or completely inactive.
Conformation limited proteolysis in the common neurophysin‐copeptin precursor shown by trypsin‐Sepharose chromatographic proteolysis
Authors: Chauvet Jacqueline , Chauvet Marie-Thérèse , Acher Roger
Published: July 2002

The guinea pig two‐domain precursor of MSEL‐neurophysin and copeptin has been passed through a trypsin‐Sepharose column in order to mimic the enzyme processing by a membrane‐bound endopeptidase. Only two cleavages were observed located in the inter‐domain sequence (at Arg‐94 and Arg‐98), in contrast to several additional cleavages found when free neurophysin or copeptin is subjected to soluble trypsin. Because the physiological maturation involves a single cleavage at Arg‐94, both local accessibility in the precursor and narrow specificity of the enzyme are implied in the processing.
A novel cholera toxin‐sensitive G‐protein (Gc) regulating receptor‐mediated phosphoinositide signalling in human pituitary clonal cells
Authors: Lo William W.Y. , Hughes John
Published: July 2002

Recent studies have implicated that a GTP‐binding protein (G‐protein) is involved in the coupling of both CCK‐8 and muscarinic cholinergic receptors to phosphoinositidase C (PIC) in the human embryonic pituitary cell line, Flow 9000. Pretreatment of these cells with cholera toxin, but not pertussis toxin, inhibited the stimulation of [3H]inositol phosphate production by CCK‐8 and acetylcholine. These inhibitory effects of cholera toxin could not be reproduced by treating the cells with the B‐subunit of cholera toxin or cAMP‐generating agents such as forskolin. These data suggest the presence of a novel Gc protein which is responsible for receptor‐PIC coupling in Flow 9000 cells.
Reconstitution of isolated Rieske Fe‐S protein into a Rieske‐depleted cytochromeb6‐ƒ complex
Authors: Adam Zach , Malkin Richard
Published: July 2002

The Rieske Fe‐S protein can be isolated from the cytochromeb6‐ƒ complex by means of chromatography on a hydroxyapatite column in the presence of detergent. Depletion of the cytochrome complex from the Rieske protein results in the loss of oxidoreductase activity, as well as the ability to reduce cytochromeb6. The Rieske Fe‐S protein can be reconstituted into the Rieske‐depleted complex by removal of the Triton X‐100 molecules associated with the protein fractions, and their substitution by lipids. Upon reconstitution the complex is reactivated, and the role of the Rieske Fe‐S protein in the reduction of both plastocyanin and cytochromeb6can be demonstrated.
Low temperature spectrophotometry on the photoreaction cycle of sensory rhodopsin
Authors: Ariki Masahiro , Shichida Yoshinori , Yoshizawa Tôru
Published: July 2002

The photoreaction of sensory rhodopsin (sR) was studied by using low temperature spectrophotometry. Upon irradiation of sR590at temperatures ranging from −30 to −60°C, sR373, a photointermediate was produced without any preceding photoproducts. The formation of sR373was temperature‐dependent, and an Arrhenius plot gave an activation energy of 15.7 kcal/mol, suggesting that there might be a potential barrier in the excited state of sR590.
A simple method for delineating well‐defined and variable regions in protein structures determined from interproton distance data
Authors: Nilges Michael , Clore G.Marius , Gronenborn Angela M.
Published: July 2002

A simple method is described for identifying well‐defined regions in a set of protein structures calculated from experimental interproton distance restraints. Two different functions, one based on the mean global rms difference, the other on the distance variation between equivalent atoms in different residues, are used to distinguish ‘variable’ from ‘well‐defined’ regions. These functions are calculated in an iterative manner. The method is also capable of identifying several locally well‐defined regions whose relative positions are not well‐defined globally.
C‐terminal labelling of β‐casein
Authors: Carles Christophe , Gueguen Paul , Ribadeau-Dumas Bruno
Published: July 2002

This paper is the first to report specific labelling of a native protein at its C‐terminal end by carboxypeptidase Y‐catalyzed transpeptidation between β‐casein and tritiated Phe amide. A tryptic digest of the radiolabelled protein was resolved by reversed‐phase HPLC and a single labelled peptide was isolated therefrom. Sequence determination and FAB mass spectrometry showed that the last 2 residues (Val‐209, Ile‐208) of β‐casein had been deleted and Ile 207 substituted by Phe, deamidation presumably occurring after transpeptidation. Identical results were obtained by transpeptidating the isolated C‐terminal tryptic heptapeptide (203–209) of native β‐casein.
Design and synthesis of metabolically stable atrial natriuretic factor analogs
Authors: Berman Judd M. , Chen Teng-Man , Sargent Roger , Buck Stephen H. , Shea Phillip , Heminger Eileen F. , Broersma Robert J.
Published: July 2002

Two analogs of rat atrial natriuretic factor, rANF7–28‐NH2 and [Mpr7, Ala2O,D‐Arg27]rANF7–27‐NH2 were prepared by the solid‐phase method. These peptides had 2‐fold and 7‐fold less affinity, respectively, than rANF1–28 in binding to membranes prepared from cultured aortic smooth muscle cells, and both peptides were 5‐fold less potent than rANF1–28 in relaxing serotonin‐contracted rabbit aortic rings. rANF7–28‐NH2 was rapidly degraded by rat kidney homogenates but [Mpr7Ala2O,D‐Arg27]rANF7–27‐NH2 had enhanced stability against rat kidney homogenate degradation. However, this in vitro stability did not translate into an extended duration of action in vivo.
Colchicine acts as a progression factor to initiate DNA synthesis in quiescent Balb/c 3T3 cells
Authors: Shinohara Yukiko , Nishida Eisuke , Sakai Hikoichi
Published: July 2002

In quiescent Balb/c 3T3 cells, competence factors such as 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and platelet‐derived growth factor (PDGF) synergize with progression factors such as insulin to initiate DNA synthesis.In this study, we found that colchicine, a microtubule‐disrupting agent, acted synergistically with TPA, but not with insulin, to induce the maximal stimulation of DNA synthesis. Colchicine also synergized with PDGF in the presence of epidermal growth factor to elicit nearly the optimal induction of DNA synthesis. Moreover, it acted synergistically with fibroblast growth factor, another competence factor. These results suggest that colchicine acts as a progression factor like insulin in quiescent Balb/c 3T3 cells.
Import of precursor proteins into Vicia faba mitochondria
Authors: Whelan James , Dolan Liam , Harmey Matthew A.
Published: July 2002

The ADP/ATP translocator from Vicia faba and β F1‐ATPase from Neurospora crassa were imported into plant mitochondria from a wheat germ and a rabbit reticulocyte translation system. Incubation of Vicia mitochondria in postribosomal supernatants of the translation reactions resulted in import of the ADP/ATP translocator. Precursor to the β‐subunit of the F1‐ATPase from N. crassa was imported and processed by the Vicia mitochondria. Import into a protease‐protected location was abolished by the inclusion of apyrase or valinomycin in the import mixtures. The Neurospora β‐subunit precursor showed post‐maturation processing to a protein with a molecular mass equivalent to that of the plant β‐subunit.
S‐100b protein regulates the activity of skeletal muscle adenylate cyclase in vitro
Authors: Fanò G. , Fulle S. , Torre G.Della , Giambanco I. , Aisa M.C. , Donato R. , Calissano P.
Published: July 2002

We have investigated the effect of the b isoform of S‐100 proteins on adenylate cyclase activity of rat skeletal muscle. S‐100b inhibits the adenylate cyclase activity in the presence of Mg2+ (5.0–50 mM), while it activates the same enzyme in the presence of Ca2+ (0.1–1.0 mM) dose‐dependently in both cases. S‐100b counteracts the stimulatory effect of NaF on adenylate cyclase in the presence of Mg2+ and the inhibitory effect of RMI 12330 A in the presence of Ca2+.
Murine mRNA for the β‐subunit of integrin is increased in BALB/c‐3T3 cells entering the G1 phase from the G0 state
Authors: Tominaga Shin-ichi
Published: July 2002

The amount of murine mRNA for the β‐subunit of integrin is enriched 6‐fold when BALB/c‐3T3 cells traverse from the G0 state to the G1 phase, whereas it remains at the basal level when the cells are growing continuously. The peak of its appearance is at 10 h after serum stimulation. The increase in integrin mRNA at a specific point in cell proliferation may be correlated with growth‐signal transduction.
Electron transfer from A−1to an iron‐sulfur center witht= 200 ns at room temperature in photosystem I Characterization by flash absorption spectroscopy
Authors: Brettel K.
Published: July 2002

Forward electron transfer in intact photosystem I particles fromSynechococcussp. at room temperature has been studied by flash absorption spectroscopy with a time resolution of 5 ns. A kinetic phase witht= 200 ns was resolved and characterized by its absorption difference spectrum between 325 and 495 nm. This phase is attributed to electron transfer from the reduced redox center A1to an iron‐sulfur center, probably Fx. The difference spectrum for the reduction of A1is evaluated and the previously proposed identification of A1with vitamin K1is discussed.
Iron storage in Saccharomyces cerevisiae
Authors: Raguzzi Fabio , Lesuisse Emmanuel , Crichton Robert R.
Published: July 2002

A ferritin‐like molecule was purified from iron‐loaded cells of Saccharomyces cerevisiae, but its iron content was very low and was not representative of the cellular iron content. A study of the intracellular distribution of iron has shown that the vacuoles are involved in the storage of iron in the yeast cell. Moreover, it seems that this vacuolar iron can be further utilised by the cells for iron‐requiring processes such as mitochondriogenesis.
The first crystallization of a vanadium‐dependent peroxidase
Authors: Müller-Fahrnow A. , Hinrichs W. , Saenger W. , Vilter H.
Published: July 2002

Single crystals of a vanadium‐containing peroxidase from the brown alga Ascophyllum nodosum have been grown by the vapour diffusion technique using polyethylene glycol 6000 along with sodium chloride as precipitant. The crystals belong to the monoclinic space group P21. X‐ray diffraction extends to at least 2.4 Å. The cell dimensions a = 173.0, b = 164.9, c=68.5 Å, β=94.5° indicate that there are four molecules of 100 kDa per asymmetric unit, suggesting that the native enzyme might occur as a tetramer.
Bicarbonate binding and the properties of photosystem II electron acceptors
Authors: Nugent Jonathan H.A. , Corrie Andrew R. , Demetriou Christalla , Evans Michael C.W. , Lockett Christopher J.
Published: July 2002

We have investigated the quinone‐binding region of PS II using exchange reactions to replace key components. Bicarbonate was replaced by formate and QBby exogenous quinones or herbicide. The effects of these changes were monitored using the ESR signals from the QAiron‐semiquinone and Fe3+non‐haem iron components. We observed that QBbinding caused characteristic changes in the Q−AESR signal and confirmed that the characteristics of Q−Adepend on bicarbonate binding. The non‐haem iron was oxidised only under conditions where bicarbonate was bound. The characteristics of bicarbonate binding were observed inPhormidium laminosumPS II, showing that the bicarbonate effect occurs in cyanobacteria. The results support a hypothesis which gives a central role to bicarbonate in providing the conditions for electron transfer in both cyanobacteria and higher plant PS II. It is suggested that bicarbonate binds at or close to the non‐haem iron and influences the characteristics of QA, QBand the non‐haem iron.
Frictional resistance to motions of bimane‐labelled spinach calmodulin in response to ligand binding
Authors: Yuan Shixing , Haug Alfred
Published: July 2002

The single cysteinyl residue 26 of spinach calmodulin was labelled with the thiol‐specific bimane fluorescence probe. Following application of stoichiometric quantities of Ca2+ or aluminum ions to the protein, temperature‐dependent fluorescence changes (anisotropy, lifetime) could be monitored via the label. From these data the Y function could be constructed which, as a function of temperature, seems to consist of two linear regions which intersect at the critical temperature, T
c. From the Y function the thermal coefficient, b(T), of the frictional resistance to fluorophore rotation could be determined. b(T) was dependent on the type and stoichiometry of the ligand(s) bound to calmodulin. Changes of the thermal coefficient apparently resulted in part from ligand‐triggered structural pertubations transmitted over a considerable distance to calmodulin region I, the site of the fluorophore.
The crystal structure of the three‐iron ferredoxin II from Desulfovibrio gigas
Authors: Kissinger C.R. , Adman E.T. , Sieker L.C. , Jensen L.H. , LeGall J.
Published: July 2002

The crystal structure of oxidized ferredoxin II from the sulfate‐reducing bacterium Desulfovibrio gigas has been determined and refined at 1.7 Å resolution. The folding of the polypeptide chain is similar to that of the 2[4Fe‐4S] ferredoxin in Peptococcus aerogenes, except for an extended helical segment near the C‐terminus. The single [3Fe‐4S] cluster in D. gigas is similar to a [4Fe‐4S] cluster, but lacks one Fe atom and is coordinated to Cys‐8, ‐14 and ‐50. The side chain of Cys‐11 is not bound to the cluster, but is rotated toward the solvent and modified by some, as yet undetermined, chemical group. Cys‐18 and Cys‐42 form a disulfide bridge. A previously undetected extra amino acid is found after residue 55.
The surface potential on the purple membrane measured using a modified bacteriorhodopsin chromophore as the spectroscopic probe
Authors: Ehrenberg Benjamin , Ebrey Thomas G. , Friedman Noga , Sheves Mordechai
Published: July 2002

The surface potential of the purple membrane was measured by a novel method by using an artificial bacteriorhodopsin whose chromophore was 13‐CF3 retinal instead of retinal. When attached to the apoprotein by a Schiff base, the intrinsic pK of the 13‐CF3 chromophore is around 7.3. The apparent pK of this pigment depends on the surface potential and thus on the electrolyte concentration. This allowed us to determine the surface charge density using the Gouy‐Chapman equation. The surface charge density was found to be −1.65 ± 0.15 × 10−3 electronic charges per Å2 or about 2 negative charges/bacteriorhodopsin. This large value for the surface potential probably explains both part of the strong apparent association of divalent cations with the membrane and the effect of low salt concentrations on light‐induced proton release from the purple membrane.
Retinoic acid enhances VIP receptor expression and responsiveness in human neuroblastoma cell, SH‐SY5Y
Authors: Waschek James A. , Muller Jean-Marc , Duan Dah-Shuhn , Sadée Wolfgang
Published: July 2002

Retinoic acid (RA) induces partial differentiation of neuroblastoma (NB) cells in vitro. In the human NB line, SH‐SY5Y (a neuroblastic subclone of SK‐N‐SH), RA was previously shown to enhance the stimulatory (PGE1) and inhibitory (opioid) regulation of adenylyl cyclase. Since these cells are also sensitive to cAMP stimulation by vasoactive intestinal peptide (VIP), we have tested the effects of RA on VIP receptor expression and function. Pretreatment of SH‐SY5Y cells with 10 μM RA over 6 days dramatically increased VIP receptor number from ∼3 000 to ∼70 000 sites per cell and enhanced threefold the cAMP accumulation after external VIP addition, while VIP immunoreactive content in the cells increased 2–3‐fold. In the light of the recently proposed autocrine function of VIP in this cell lineage, the strong enhancement of the VIP system may contribute to the differentiation effects of RA.
Possible role of some groups in the structure and function of HIV‐1 protease as revealed by molecular modeling studies
Authors: Pechik I.V. , Gustchina A.E. , Andreeva N.S. , Fedorov A.A.
Published: July 2002

Retroviral proteases belong to the class of aspartic proteases. A molecular model of HIV‐1 protease has been built on the basis of the consensus template specific for the domains of these enzymes. The template region comprises more than a half of the HIV‐1 protease monomer structure, it includes the active site, formed at the junction of the two monomers, binding pockets of the enzyme, and some other molecular segments. These regions can be more conveniently described than other parts of the structure. Some properties of the HIV‐1 protease molecule are discussed, as well as of probable inhibitors. The properties of the model structure are in good agreement with the recent results of crystallographic studies of Rous sarcoma virus protease.
Transport of malate and chloride into barley mesophyll vacuoles Different carriers are involved
Authors: Martinoia Enrico , Vogt Esther , Amrhein Nikolaus
Published: July 2002

Transport of malate and chloride across the tonoplast of barley mesophyll vacuoles has been compared. Whereas malate inhibited chloride uptake, 1,2,3‐benzenetricarboxylic acid, a potent inhibitor of malate uptake which is assumed not to cross the tonoplast, was ineffective. In contrast to chloride uptake, malate uptake was inactivated by pyridoxal phosphate and phenylglyoxal. The malate transport system could be protected against pyridoxal phosphate when a substrate such as 1,2,3‐benzenetricarboxylic acid was present in the preincubation medium. It is concluded that the transfer of malate and chloride across the tonoplast is mediated by different systems.
Crystallization of blood coagulation factor XIII by an automated procedure
Authors: Hilgenfeld Rolf , Liesum Alexander , Storm Rüdiger , Metzner Hubert J. , Karges Hermann E.
Published: July 2002

Both recombinant blood coagulation factor XIII a‐chain and factor XIII isolated from human placenta have been crystallized using a novel robotic system for the automatic screening of crystallization conditions. The monoclinic and orthorhombic crystals obtained are suitable for X‐ray analysis.
Neurospora crassa cDNA clones coding for a new member of the ras protein family
Authors: Altschuler Daniel L. , Muro Andres , Schijman Alejandro , Almonacid Fernando Bravo , Torres Hector N.
Published: July 2002

A new member of the ras gene family was characterized from Neurospora crassa cDNA libraries. The clone designated NC‐ras codes for a polypeptide containing 213 amino acids (M
r 24000). This polypeptide is 84% homologous to the H‐ras‐1 domain comprising the first 80 amino acids and 60% homologous to the next 84 residues. The NC‐ras polypeptide contains all the well‐known sequences involved in the interaction with GTP/GDP, the recognition of the Y13‐259 neutralizing antibody, the ‘effector site’ for interaction with GAP proteins, and the CAAX acylation motif in the COOH‐terminal.
Generation of inositol phosphates, cytosolic Ca2+ and secretion of noradrenaline in PC12 cells treated with glutamate
Authors: Kurozumi Kouichi , Murayama Toshihiko , Nomura Yasuyuki
Published: July 2002

Glutamate transiently stimulated rat pheochromocytoma PC12 cells and caused an inositol trisphosphate formation and an increase in levels of Ca+ in the cytosol. The rank order of potency of glutamate >
N‐methyl‐D‐aspartate (NMDA) > kainate = quisqualate is characteristic of an interaction with NMDA receptors. The effect of glutamate on inositol trisphosphate formation disappeared in a low Mg2+ buffer and was not blocked by DL‐2‐amino‐5‐phosphonovalerate, an antagonist for NMDA receptors coupled to ion channels. Although glutamate failed to stimulate noradrenaline secretion, glutamate enhanced the effect of bradykinin, but not of Ca ionophore A23187, or KC1. These results suggest the existence of metabotropic glutamate receptors, different from previously reported receptors, in PC12 cells.
Purification and characterization of CMP‐NeuAc:GM1 (Galβ1‐4Ga1NAc) α2‐3 sialyltransferase from rat brain
Authors: Gu Tian-Jue , Gu Xin-Bin , Ariga Toshio , Yu Robert K.
Published: July 2002

A CMP‐NeuAc:GM1 α2‐3sialyltransferase (GD1a synthase, 2.4.99.2) has been purified from the Triton extract of rat brain. The enzyme was purified and resolved by affinity chromatography on CDP‐Sepharose column by a linear NaCl gradient elution. Final purification was achieved by elution from a ‘GM1‐acid’‐Sepharose column. SDS‐PAGE of the enzyme revealed a single protein band with an apparent M
r 44 kDa. It catalyzed specifically the sialylation of GD1b, GM1 and asialo‐GM1. Enzyme products were identified by TLC in three different solvent systems, The K
m value for GM1 was 7.5 × 10−2 M, and for CMP‐NeuAc it was 6.5 × 10−5 M.
Effects of neutrophil adherence on the characteristics of receptors for tumor necrosis factor‐α
Authors: Chamba Anita , Stockley Robert A. , Burnett David
Published: July 2002

Human recombinant [125]TNF‐α was incubated with non‐adherent human neutrophils, cells adherent to fibronectin‐coated plastic, or adherent cells scraped into suspension (post‐adherent). Binding of TNF to all cells increased with doses of added TNF but adherent cells little TNF, Binding of TNF by post‐adherent cells was greater than when adherent, but still significantly less than of non‐adhered neutrophils, suggesting that TNF receptors were relocated on the adherent surface of neutrophils. Scatchard analysis showed that adherent cells expressed significantly fewer TNF receptors, but of higher affinity, then non‐adherent cells. The results suggest that altered expression of TNF receptors might contribute to the differential effects of TNF on adherent and non‐adherent neutrophils.
Increased responsiveness of rat mast cells to compound 48/80 due to removal of extracellular magnesium Effects of ouabain and EGTA
Authors: Bertelsen Henrik , Johansen Torben
Published: July 2002

A decreased secretory response of mast cells to compound 40/80 (12% of control value) after preincubation of the cells with magnesium but without calcium was partially restored by removal of magnesium. EGTA (10 μM) blocked the restoration and decreased the restored secretory activity again, while this was further increased by ouabain (1 mM). Furthermore, ouabain completely restored the decreased secretion (50% of control value) due to preincubation without the divalent cations. This may indicate that magnesium influences a pool of cellular calcium that is involved in the stimulus‐secretion coupling and is available to EGTA, and ouabain did not counteract the inhibitory mechanism or magnesium.
Modification of α‐amylase from Bacillus licheniformis by the polyaldehyde derived from β‐cyclodextrine and α‐amylase thermostability
Authors: Morand Philippe , Biellmann Jean-François
Published: July 2002

The cleavage of β‐cyclodextrine by sodium periodate at the seven 2–3 diols of the glucose unit gives rise to the polyaldehyde 1, used to modify α‐amylase. The reductive modification of α‐amylase from Bacillus licheniformis reduced the number of reactive lysine groups from 8 to 3.5 per mol of enzyme with an activity loss of 25% and increased the half‐life at 80°C from 4.7 to 7.0 minutes.
Interleukin‐4 inhibits the differentiation of mouse myeloid leukemia M1 cells induced by dexamethasone, D‐factor/leukemia inhibitory factor and interleukin‐6, but not by 1α,25‐dihydroxyvitamin D3
Authors: Kasukabe Takashi , Okabe-Kado Junko , Honma Yoshio , Hozumi Motoo
Published: July 2002

The effects of interleukin‐4(IL‐4) on the growth and differentiation of mouse myeloid leukemia M1 cells induced by various differentiation inducers were investigated. IL‐4 alone did not have any significant effect on the growth or differentiation of M1 cells, but inhibited their differentiation induced by dexamethasone, D‐factor/leukemia inhibitory factor, or interleukin 6. IL‐4 also restored the proliferation or M1 cells after growth inhibition during their induction of differentiation by inducers. In contrast, IL‐4 enhanced inhibition of growth and induction of differentiation of M1 cells by 1α,25‐dihydroxyvitamin D3. These results indicate that modulation or differentiation of M1 cells by IL‐4 depends on the differentiation inducer.
Relationship between intracellular pH changes, activation of protein kinase C and NADPH oxidase in macrophages
Authors: Dieter Peter
Published: July 2002

Activation of the superoxide‐generating NADPH oxidase by phorbol ester or zymosan induced a cytoplasmic acidification when liver macrophages were incubated in sodium free media or in the presence of amiloride. Staurosporine or desensitization of protein kinase C inhibited phorbol ester‐and zymosan‐induced pH changes and generation of superoxide. The intracellular pH remained unchanged in cells incubated in physiological sodium media. Ionomycin and arachidonic acid did not induce a change in intracellular pH or a generation of superoxide. Fluoride, which has been shown to induce a translocation of protein kinase C in these cells, did not elicit superoxide generation but induced a decrease in intracellular pH. These experiments support (1) a role of the Na+/H+ antiporter in macrophages as a metabolic regulator of intracellular pH upon stimulation of the superoxide‐generating NADPH oxidase, and (2) suggest an involvement of protein kinase C in this process.
Inhibition of chloroplast CF1‐ATPase by vanadate
Authors: Carmeli C. , Tadmor O. , Lifshitz Y. , Ophir R. , Carmeli S.
Published: July 2002

Inhibition of ATPase activity by vanadate, having K½ of 0.5 mM, was demonstrated in the CF1‐ATPase. The Ca2+‐dependent ATPase activity of the isolated enzyme was inhibited in an allosteric manner by vanadate with a Hill coefficient of 3.19 ± 0.6. Vanadate also inhibited ATPase and Pi—ATP exchange activities of the chloroplast membrane‐bound enzyme. Using 51V NMR it was demonstrated that ATP caused partial release of about 1.87 equivalents while ADP caused additional binding of approximately 1.46 equivalents of vanadate, when added to a solution containing CF1 equilibrated with vanadate. The relevance of these results to a possible involvement of a pentacovalent phosphate as transition state intermediate in the hydrolysis of ATP by CF1‐ATPase is discussed.
(Model) studies on vanadate‐dependent bromo/iodoperoxidase from Ascophyllum nodosum VO2+ is not incorporated into the active site
Authors: Knüttel Karlheinz , Müller Achim , Rehder Dieter , Vilter Hans , Wittneben Volker
Published: July 2002

Vanadate‐dependent peroxidase A.n.I. the main isoenzyme (M
r = 100 kDa) from the seaweed. Ascophyllum nodosum, contains 2 V per enzyme molecule (as shown by ICP‐MS metal analysis) after complete reconstitution with vanadate (V), possibly distributed in a 1:1 ratio between the surface and active site. VO2+ is only weakly associated to the surface of A,n.I. There is no transport channel for VO2+. The EPR spectrum of the reduced holoenzyme is unisotropic (axial) already at room temperature, with EPR parameters similar to those of VO2+ complexes of small model peptides such as Ala‐His, Gly‐Tyr, Gly‐Ser, Gly‐Glu, Ser‐Gly and Phe‐Glu. The complex formation between Ala‐His and H2VO−
4 in water has also been investigated (by 51V NMR); the formation constant at pH 7.2 amounts to 266(28) M−1.
Synthesis and characterization of a 29‐amino acid residue DNA‐binding peptide derived from α/β‐type small, acid‐soluble spore proteins (SASP) of bacteria
Authors: Rao Hai , Mohr Scott C. , Fairhead Heather , Setlow Peter
Published: July 2002

A 29‐amino acid residue peptide (SASP‐peptide) derived from the sequence of the putative DNA‐contacting portion at the carboxyl terminus of an α/β‐type small, acid‐soluble spore protein (SASP) of Bacillus subillis has been synthesized by automated solid‐phase methods and tested for its ability to interact with DNA. Circular dichroism (CD) spectroscopy reveals an interaction between this SASP‐peptidc and DNA, both by an increase in α‐helix content of the peplide (which alone has a mostly random conformation) and by enhancement of the 275‐nm CD band of the DNA. In contrast to results with intact α/β‐type SASP, however, the peptide does not induce a B → A cenformational transition in the DNA. The SASP‐peptide also binds to poly(dG)·poly(dC) and prtects this polynucleotide against DNase I digestion and UV light‐induced cylosine dimer formation, parallel to Findings made previously with native α/β‐type SASP. The results confirm the hypothesis that the carboxyl‐terminal region of the α/β‐type SASP directly contacts DNA and possesses some, but not all, or the functional characteristics of the intact molecule.
Activation of peritoneal macrophages during the prediabetic phase in low‐dose streptozotocin‐treated mice
Authors: Andrade Josefa , Conde Manuel , Sobrino Francisco , Bedoya Francisco J.
Published: July 2002

Glucose metabolism and the production of O2
− and NO2
− have been studied in peritoneal macrophages from mice injected with 5 subdiabetogenic doses of streptozotocin. On day 12 after beginning of the treatment, peritoneal macrophages produced significantly higher amounts of lactate than macrophages from control mice. In addition, NO2
− release and phorbol ester‐induced O2
− production were significantly augmented in macrophages from streptozotocin‐treated mice. γ‐Interferon induced in a dose‐dependent manner the activity of NO synthase only in macrophages from streptozotocin‐treated mice. These data show for the first time that peritoneal macrophages from streptozotocin‐treated mice are activated and produce effector molecules such as O2
− and NO which could participate in the destruction of pancreatic islets.
Disparate contributions of Tyr10 and Tyr109 to fluorescence intensity of rabbit skeletal muscle troponin C identified using a genetically engineered mutant
Authors: Keleti David , Rao Venu G. , Su Hong , Akella Arvind B. , Ding Xiao-Ling , Gulati Jagdish
Published: July 2002

Intrinsic tyrosines, as monitored by fluorescence spectroscopy, are sensitive reporters of local, Ca2+‐induced conformational changes in troponin C (TnC). Rabbit skeletal TnC contains two tyrosines (Y10 in the N‐helix, and Y109 in site 3 in the C‐terminal domain) in distinct microenvironments: their individual contributions to total fluorescence intensity are elucidated here utilizing bacterially synthesized rabbit skeletal TnC (sTnC4) and a genetically engineered variant, termed 109YF, lacking one of the tyrosines (Y109 replaced with F109). The steady‐state fluorescence emission spectra following excitation at 280 nm were recorded in EGTA (Ca2+‐free) and Ca2+‐saturated (pCa4) solutions. For the wild‐type sTnC4, pCa4 causes a significant (46%) increase in the peak fluorescence intensity over the value in EGTA. For the mutant 109YF, the EGTA fluorescence is only marginally affected (74% of the wild‐type F
EGTA), but interestingly the Ca2+ effect is completely suppressed (ΔF = F
pCa4 ‐ F
EGTA = 2% of the wild‐type value). These results indicate that the two tyrosines make disparate contributions to the fluorescence spectrum of wild‐type sTnC, both in the presence and absence of Ca2+; whereas Y10 in the N‐helix is dominant in Ca2+‐free solution, Y109 is the sole contributor to the Ca2+ effect. Furthermore, to explain the biphasic fluorescence response of Y109 obtained during Ca2+ titrations, the findings yield the most unequivocal evidence that Ca2+‐induced conformational changes in the trigger sites operating the contractile switch modify properties of the C‐terminal sites in TnC pari passu.
The role of the autoinhibitory domain in differential metal ion activation of calmodulin‐stimulated phosphatase
Authors: Yokoyama Noriko , Wang Jerry H.
Published: July 2002

Metal ion activators, Ni2+ and Mn2+, have been suggested to induce different conformations of calmodulin (CaM)‐stimulated phosphatase. In the present study, an autoinhibitory domain previously implicated in the conformation transition of CaM stimulation of the phosphatase is shown to participate in defining the differential metal ion activation. A proteolytic derivative of the phosphatase deleted from the autoinhibitory domain displayed CaM‐independent Mn2+‐stimulated activity which was about 4‐times that of the CaM‐stimulated activity of the native enzyme. The Ni2+‐stimulated activity of the derivative, on the other hand, retained slight CaM‐dependence, and the CaM‐stimulated activity was 90% of that of the native enzyme. A synthetic peptide corresponding to the autoinhibitory domain could inhibit the Mn2+‐stimulated activity of the phosphatase derivative by 80%, but had little effect on the Ni2+‐stimulated activity.
Structural similarities between chaperone molecules of the HSP60 and HSP70 families deduced from hydrophobic cluster analysis
Authors: Callebaut Isabelle , Catelli Maria Grazia , Portetelle Daniel , Burny Arséne , Baulieu Etienne Emile , Mornon Jean Paul
Published: July 2002

In this study, the conservation of strong structural landmarks between all the members of two chaperone families (HSP60 and HSP70) was deduced from their sequences by hydrophobic cluster analysis. On this basis, we propose that the ATP‐binding environment is maintained by a similar fold in both protein families. The observed similarities extend throughout the proteins, including both the ATPase domain and the C‐terminal substrate‐binding domain.
Flavones and flavonols at dietary levels inhibit a transformation of aryl hydrocarbon receptor induced by dioxin
Authors: Ashida Hitoshi , Fukuda Itsuko , Yamashita Takatoshi , Kanazawa Kazuki
Published: July 2002

Dioxins invade the body mainly through the diet, and produce toxicity through the transformation of aryl hydrocarbon receptor (AhR). An inhibitor of the transformation should therefore protect against the toxicity and ideally be part of the diet. We examined flavonoids ubiquitously expressed in plant foods as one of the best candidates, and found that the subclasses flavones and flavonols suppressed antagonistically the transformation of AhR induced by 1 nM of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin, without exhibiting agonistic effects that transform AhR. The antagonistic IC50 values ranged from 0.14 to 10 μM, close to the physiological levels in human.
Nitric oxide protects Cu,Zn‐superoxide dismutase from hydrogen peroxide‐induced inactivation
Authors: Kim Yu Shin , Han Sanghwa
Published: July 2002

Reaction of Cu,Zn‐superoxide dismutase (SOD1) and hydrogen peroxide generates a putative oxidant SOD‐Cu2+‐OH that can inactivate the enzyme and oxidize 5,5′‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) to DMPO‐OH. In the presence of nitric oxide (NO), the SOD1/H2O2system is known to produce peroxynitrite (ONOO−). In contrast to the proposed cytotoxicity of NO conferred by ONOO−, we report here a protective role of NO in the H2O2‐induced inactivation of SOD1. In a dose‐dependent manner, NO suppressed formation of DMPO‐OH and inactivation of the enzyme. Fragmentation of the enzyme was not affected by NO. Bicarbonate retarded formation of ONOO−, suggesting that NO competes with bicarbonate for the oxidant SOD‐Cu2+‐OH. We propose that NO protects SOD1 from H2O2‐induced inactivation by reducing SOD‐Cu2+‐OH to the active SOD‐Cu2+with concomitant production of NO+which reacts with H2O2to give ONOO−.
Identification and characterization of functional domains in a mixed lineage kinase LZK
Authors: Ikeda Atsushi , Masaki Megumi , Kozutsumi Yasunori , Oka Shogo , Kawasaki Toshisuke
Published: July 2002

The mixed lineage kinase (MLK) family is a recently described protein kinase family. The MLKs contain a kinase domain followed by a dual leucine zipper‐like motif. We previously reported the molecular cloning of LZK (eucine ipper‐bearing inase), a novel MLK, and that LZK activated the c‐Jun NH2 terminal kinase (JNK)/stress‐activated protein kinase (SAPK) pathway through MKK7 in cells. Here, we reveal that LZK forms dimers/oligomers through its dual leucine zipper‐like motif, and that this is necessary for activation of the JNK/SAPK pathway. We also identify the C‐terminal functional region of LZK, which is indispensable for the activation of SEK1, but not that of MKK7.
Different susceptibility to oxidation of proline and arginine residues of apolipoprotein B‐100 among subspecies of low density lipoproteins
Authors: Pietzsch Jens , Julius Ulrich
Published: July 2002

γ‐Glutamyl semialdehyde is a primary oxidation product of apolipoprotein (apo) B‐100 proline (Pro) and arginine (Arg) side chain residues. By reduction γ‐glutamyl semialdehyde forms 5‐hydroxy‐2‐aminovaleric acid (HAVA). Here we describe the application of sensitive and specific HAVA measurement to characterize the formation of γ‐glutamyl semialdehyde in several domains of apoB‐100 in LDL1 (Sf 7–12) and LDL2 (Sf 0–7) subfractions subjected to oxidative damage in the presence of iron in vitro. Results suggest that susceptibility of apoB‐100 Pro and Arg residues toward oxygen radicals drastically changes along the lipoprotein metabolic cascade.
NaCl‐activated nucleoside diphosphate kinase from extremely halophilic archaeon, Halobacterium salinarum, maintains native conformation without salt
Authors: Ishibashi Matsujiro , Tokunaga Hiroko , Hiratsuka Kazushi , Yonezawa Yasushi , Tsurumaru Hirohito , Arakawa Tsutomu , Tokunaga Masao
Published: July 2002

Enzymes from extremely halophilic archaea are readily denatured in the absence of a high salt concentration. However, we have observed here that a nucleoside diphosphate kinase prepared from Halobacterium salinarum was active and stable in the absence of salt, though it has the amino acid composition characteristic of halophilic enzymes. Recombinant nucleoside diphosphate kinase expressed in Escherichia coli requires salt for activation in vitro, but once it acquires the proper folding, it no longer requires the presence of salts for its activity and stability.
Chemical modification of α2‐macroglobulin to generate derivatives that bind transforming growth factor‐β with increased affinity
Authors: Webb Donna J. , Gonias Steven L.
Published: July 2002

α2‐Macroglobulin (α2M) binds a number of cytokines, including transforming growth factor‐β1 (TGF‐β1) and TGF‐β2. The affinity of these interactions depends on the α2M conformation. In this investigation, we treated human α2M with cis‐dichlorodiammineplatinum (II) (cis‐Pt), a crosslinking reagent that partially ‘locks’ the α2M conformation, and then with methylamine to generate a preparation (α2M‐P/M) consisting of stable α2M conformational intermediates. α2M‐P/M bound TGF‐β1 and TGF‐β2 with higher affinity than any other form of α2M studied to date. The equilibrium dissociation constants were 14 and 2 nM for TGF‐β1 and TGF‐β2, respectively. α2M‐P/M, at 100 nM, neutralized the activity of TGF‐β1 by about 75% in an endothelial cell proliferation assay. The equivalent concentration of native α2M or methylamine‐modified α2M had no effect. These studies demonstrate that the potential of α2M as a cytokine carrier and neutralizer may not be fully realized in either the native or completely activated conformations.
N,N‐Dimethylsphingosine 1‐phosphate activates human platelets
Authors: Yatomi Yutaka , Yamamura Soichiro , Ruan Fuqiang , Kume Shoji , Ozaki Yukio , Igarashi Yasuyuki
Published: July 2002

We recently reported that N,N‐dimethylsphingosine 1‐phosphate (DMS‐1‐P) can be formed from N,N‐dimethylsphingosine (DMS) in activated platelets [Y. Yatomi et al., Biochem. Biophys. Res. Commun. 231 (1997) 848–851]. In this study, we synthesized, for the first time, DMS‐1‐P and examined the functional effects of DMS‐1‐P and its related sphingolipids on platelets. Although exogenous DMS was inactive, its phosphorylated derivative, DMS‐1‐P, induced platelet intracellular Ca2+ mobilization and shape change, but not aggregation or release reactions. Since sphingosine 1‐phosphate (Sph‐1‐P) is structurally related to DMS‐1‐P and activates platelets more strongly than DMS‐1‐P, a competitive binding experiment for [3H]Sph‐1‐P was performed using DMS‐1‐P. DMS‐1‐P reduced the binding of [3H]Sph‐1‐P to platelets almost as much as unlabeled Sph‐1‐P did. These results suggest that DMS‐1‐P activates platelets via an interaction with a platelet surface receptor for Sph‐1‐P.
Analysis of the activation state of α4β1 integrin in human B cell lines derived from myeloma, leukemia or lymphoma
Authors: Garcı́a-Gila Mercedes , Cabañas Carlos , Garcı́a-Pardo Angeles
Published: July 2002

Myeloma cells specifically localize in the bone marrow and rarely circulate in blood. To study whether this immobilization could be partially explained by the presence of constitutively activated integrins, particularly α4β1, we used the activation reporter HUTS‐21 anti‐β1 mAb. These analyses showed that β1 integrins on myeloma cells were moderately active and could be upregulated similarly to integrins on lymphoma or leukemia cells. Myeloma cells were also tested for their ability to attach to RGD‐containing fibronectin fragments, a property of activated (but not resting) α4β1. Two cell lines adhered to these fragments and this was inhibited by anti‐α5 but not by anti‐α4 mAbs. These results show that myeloma cells bear low/moderately active α4β1 and support the notion that multiple interactions contribute to their localization in the bone marrow.
A phosphatase activity in Xenopus oocyte extracts preferentially dephosphorylates the MPM‐2 epitope
Authors: Che Shaoli , Wu Weiguo , Nelman-Gonzalez Mayra , Stukenberg Todd , Clark Richard , Kuang Jian
Published: July 2002

MPM‐2 antigens are a large family of mitotic phosphoproteins that contain similar phosphoepitopes recognized by the anti‐phosphoepitope antibody MPM‐2 (MPM‐2 epitopes). These proteins are phosphorylated during M phase induction and dephosphorylated from the onset of anaphase through interphase. Since biochemical characterization of the MPM‐2 epitope phosphatase requires a specific assay for its activity, we tested different methods for measurement of the MPM‐2 epitope phosphatase activity in crude cell lysates. First, an ELISA‐based assay was designed that measured the phosphatase‐induced reduction of the MPM‐2 reactivity in crude M phase cell lysates. Using this assay to follow the phosphatase activity during sequential chromatography of Xenopus oocyte extracts, one predominant peak of phosphatase activity was detected which was separated from the majority of PP1 and PP2A activities. This phosphatase activity dephosphorylated the MPM‐2 epitope on multiple MPM‐2 antigens. The second method measured dephosphorylation of cdc25, a known MPM‐2 antigen. Two major peaks of cdc25 dephosphorylating activities were detected during the sequential chromatography, one that copurified with the major peak of MPM‐2 epitope phosphatase activity, and the other with the major peak of PP2A activity. Finally, we examined whether GST‐MPM2, a fusion protein between glutathione S‐transferase and a 19‐residue peptide that contained two representative MPM‐2 epitope sequences, could be dephosphorylated efficiently and specifically by the major MPM‐2 epitope phosphatase activity in Xenopus oocyte extracts. Neither the crude extract nor the partially purified MPM‐2 epitope phosphatase activity efficiently dephosphorylated the MPM‐2 epitope on GST‐MPM2. These results demonstrate that the ELISA‐based assay preferentially detects the MPM‐2 epitope phosphatase activity in crude cell lysates which may represent a physiological MPM‐2 epitope phosphatase.
Expression and regulation of phospholipase D in the human keratinocyte cell line HaCaT
Authors: Müller-Wieprecht Verena , Riebeling Christian , Alexander Christian , Scholz Frank-Rüdiger , Höer Ariane , Wieder Thomas , Orfanos Constantin E , Geilen Christoph C
Published: July 2002

The generation of lipid second messengers via phosphatidylcholine (PC)‐specific phospholipase D (PLD) has emerged as an important step leading to transduction of extracellular signals. In the present investigation the expression of human cytosolic PLD isoenzymes in the immortalized human keratinocyte cell line HaCat was determined. At the mRNA level we found the expression of hPLD1b and for the first time in human cells also the expression of hPLD2. For further analysis of enzyme expression at the protein level, hPLD1 peptide fragments were synthesized and specific antibodies were generated (rabbit) to be used for detection of hPLD1 in Western blot experiments. Furthermore, small G‐proteins were found to be involved in the regulation of PLD activity in HaCaT cells using the guanine nucleotide analogue GTPγS.
A new system for the measurement of electrogenicity produced by ion pumps using a thin polymer film: examination of wild type bacteriorhodopsin and the D96N mutant over a wide pH range
Authors: Muneyuki Eiro , Okuno Daichi , Yoshida Masasuke , Ikai Atsushi , Arakawa Hideo
Published: July 2002

We developed a new assay system for the measurement of capacitive electric currents generated by ion pumps using the thin polymer film `Lumirror' (Toray Co., Japan). This system enables us to examine the electrogenicity of ion pumps over a wide range of experimental conditions with high reproducibility due to the mechanical and chemical stability, the high electric resistance and the high electric capacitance of the thin polymer film. Using this method, we examined the photoelectric response of wild type bacteriorhodopsin and its D96N mutant over a wide pH range (2.8–10.0). The results were explained in terms of the affinities of the proton binding sites for translocated protons. A possibility that the direction of the proton transfer from the Schiff base was influenced by the protonation/deprotonation state of the surrounding proton binding sites was suggested. We also found that this film can be used as a substrate for atomic force microscopy (AFM) samples and hence the active purple membrane was observed with AFM.