bluuebunny/crossref_metadata_2025 huggingface dataset:
Outer‐membrane porins from Gram‐negative bacteria stimulate platelet‐activating‐factor biosynthesis by cultured human endothelial cells
Authors: TUFANO Maria A. , BIANCONE Luigi , ROSSANO Fabio , CAPASSO Ciro , BARONI Adone , DE MARTINO Antonella , IORIO Eugenio L. , SILVESTRO Luigi , CAMUSSI Giovanni
Published: March 2005

Porins are a family of hydrophobic proteins located in the outer membrane of the cell wall in Gram‐negative bacteria. The effect of porins on the biosynthesis of platelet‐activating factor (PAF) by cultured human umbilical‐cord‐vein‐derived endothelial cells (HUVEC) was investigated. The results demonstrate that porins were able to induce a dose‐dependent synthesis of PAF in HUVEC. PAF, synthesized after stimulation with porins, was mainly cell associated and the synthesis peaked at 15 min, decreasing rapidly thereafter. Experiments with radiolabeled precursors demonstrated that PAF, a 1‐O‐alkyl‐2‐acetyl‐sn‐glyceryl‐3‐phosphorylcholine, was synthesized via the remodeling pathway involving the acetylation of 1‐O‐alkyl‐2‐lyso‐sn‐glyceryl‐3‐phosphorylcholine (2‐lysoPAF) generated from 1‐O‐alkyl‐2‐acyl‐sn‐glyceryl‐3‐phosphorylcholine by phospholipase‐A2 activity. The activation of phospholipase A2 in HUVEC stimulated by porins was detected by observing the mobilization of [14C]arachidonic acid. In addition, the activity of acetyl‐CoA:1‐alkyl‐sn‐glycero‐3‐phosphorylcholine 2‐O‐acetyltransferase was transiently increased in porin‐stimulated HUVEC and, after incubation with [3H]CoASAc or [3H]acetate, the [3H]acetyl group was incorporated into newly synthesized PAF. Porins, by forming transmembrane channels, induced a sustained influx of extracellular 45Ca2+ into the cytosol. The activation of PAF synthesis by porins depended on this influx rather than on intracellular calcium mobilization, since PAF synthesis did not occur in the absence of extracellular Ca2+.
Tryptophan phosphorescence as a structural probe of mitochondrial F1‐ATPase ε‐subunit
Authors: SOLAINI Giancarlo , BARACCA Alessandra , CASTELLI Giovanna Parenti , STRAMBINI Giovanni B.
Published: March 2005

We report the detection of tryptophan phosphorescence emission from the sole residue in the ε‐subunit of the bovine heart mitochondrial F1‐ATPase complex. The phosphorescence spectrum, intensity and decay kinetics have been measured over the temperature range 160–273 K. The fine structure in the phosphorescence spectrum at low temperature, with the 0–0 vibrational band centered at 411 nm, reveals the hydrophobic nature of the chromophore's environment. Both the large width of the 0–0 vibrational band and the heterogeneous decay kinetics in fluid solution emphasize the existence of multiple conformations of the ε‐subunit, structures which are rather stable as they do not interconvert in the millisecond time scale. Further, from the relatively long triplet lifetime at 273 K, it is possible to infer the existence of a tight, rigid core in the structure of the ε‐subunit. Under subunit‐dissociating conditions (6 M urea), the spectrum at 160 K undergoes a slight blue shift but since the phosphorescence lifetime, at all temperatures, is similar or longer than in the absence of dissociant, we conclude that dissociation does not lead to solvent exposure of the tryptophanyl side‐chain. This conclusion is supported by the results obtained at 273 K by dissociating F1 in the presence of 0.3 M guanidine hydrochloride. Phosphorescence lifetimes indicate that 6 M urea leads to a more compact structure of the ε‐subunit, whereas the opposite occurs when Mg‐ATP is added to nucleotide‐depleted F1. These spectroscopic changes establish unequivocally that the binding of the adenine nucleotide to the enzyme is accompanied by conformational changes involving the ε‐subunit.
Mammalian PC‐12 cell genetically engineered for human cytochrome P450 2E1 expression
Authors: MAPOLES John , BERTHOU François , ALEXANDER Alice , SIMON Francis , MÉNEZ Jean‐François
Published: March 2005

The stable expression of the human cytochrome CYP2E1 (P450 alcohol) was performed in the mammalian cell line PC‐12. This cell line expressed cytochrome b5 (58 ± 12 pmol/mg microsomal protein vs 528 ± 80 pmol/mg in microsomal human liver) and a high level of NADPH: cytochrome P450 reductase (140 ± 20 nmol · min−1· mg microsomal protein−1 vs 68 ± 48 nmol · min−1· mg−1 in microsomal human liver). An expression plasmid was constructed using the cDNA for the human CYP2E1 mRNA and the Rous sarcoma virus (RSV) promoter. This plasmid was co‐transfected with the plasmid RSVneo into PC‐12 cells. Clones were selected for resistance to the neomycin analog, G418, and then screened for expression of the CYP2E1 isozyme by testing for 6‐hydroxylation of chlorzoxazone, a specific substrate for CYP2E1. Expression of CYP2E1 was confirmed in one clone, DB‐7, by Western blot analysis and by measurement of monooxygenase activities which were not detectable in PC‐12 cells. Chlorzoxazone 6‐hydroxylation, n‐butanol oxidation and dimethylnitrosamine N‐demethylation were localized in microsomes (62, 60 and 63 pmol · min−1· mg microsomal protein−1, respectively) and were inhibited by carbon monoxide and diethyldithiocarbamate, both inhibitors of P450 enzymes. Although the level of the enzyme activities was about a tenth of that measured in human liver microsomes, CYP2E1 expressed in DB‐7 cells has catalytic competence similar to human liver CYP2E1. DB‐7 cells metabolized acetaminophen and this metabolic activation was shown to be toxic to these cells by release of lactate dehydrogenase.

Construction of recombinant cell lines expressing CYP2E1 provides a useful tool for studying the catalytic properties of this enzyme and the consequent cytotoxic effects of substrates metabolized by this enzyme.
Isomalto‐oligosaccharide‐containing lipoteichoic acid of Streptococcus sanguis
Authors: KOCHANOWSKI Bernd , FISCHER Werner , IIDA‐TANAKA Naoko , ISHIZUKA Ineo
Published: March 2005

The lipoteichoic acid of Streptococcus sanguis DSM 20567 and of DSM 20068 was isolated by phenol/water extraction and hydrophobic‐interaction chromatography. The preparations from both strains have an identical structure: a 1,3‐linked poly(glycerophosphate) chain phosphodiester‐linked to Glc‐(α1–2)Glc(α1–3)acyl2Gro as the lipid anchor. The chain is substituted with d‐alanine ester and glycosyl residues which comprise mono‐, di‐, tri‐ and tetra‐α‐d‐glucopyranosyl residues with (1–6) interglycosidic linkages. The glycosylglycerols were released with 48% (by mass) hydrofluoric acid, separated and characterized by a combination of chemical procedures and modern techniques of 1H‐NMR and 13C‐NMR spectroscopy. The α‐isomalto‐oligosaccharides add a novel motif to lipoteichoic‐acid chain substituents.

1H‐NMR and 13C‐NMR spectroscopy also provided a detailed picture of the basic glycosylated poly(1,3‐glycerophosphate) diglucosylglycerol. It proved a single unbranched chain structure, provided evidence for the chain length, the extent of glycosylation, the structure of the lipid anchor and the site of attachment of the poly(glycerophosphate) chain on the lipid anchor.

Owing to its unique glycosyl substituents the lipoteichoic acid may serve as a taxonomic marker for the redefined species S. sanguis (formerly S. sanguis type I).
An analysis of Bacillus thuringiensisδ‐endotoxin action on insect‐midgut‐membrane permeability using a light‐scattering assay
Authors: CARROLL Joe , ELLAR David J.
Published: March 2005

Changes in the membrane permeability of Manduca sexta midgut brush‐border‐membrane vesicles (BBMV) after addition of Bacillus thuringiensisδ‐endotoxins were studied using osmotic swelling experiments, volume changes being monitored as the change in 90° light scattering. Typically, control BBMV exhibited limited permeability for sucrose and salts (KCl), while being permeable for urea and glucose. The action of δ‐endotoxin was examined using proteolytically activated Cry‐IA(c) and CryIB toxins. CryIA(c) produced a marked change in the solute permeability of M. sexta BBMV, significant effects being observed at 3.75 pmol/mg BBMV. The permeability change was relatively non‐selective with cations, anions and neutral solutes all traversing the membrane to an increased extent in the presence of CryIA(c). In contrast, the CryIB toxin had no effect on BBMV permeability.
Purification and characterization of a pyranose oxidase from the basidiomycete Peniophora gigantea and chemical analyses of its reaction products
Authors: DANNEEL Hans‐Jürgen , RÖSSNER Ellen , ZEECK Axel , GIFFHORN Friedrich
Published: March 2005

A pyranose oxidase was isolated from mycelium extracts of the basidiomycete Peniophora gigantea. This enzyme was purified 104‐fold to apparent homogeneity with a yield of about 75% by steps involving fractionated ammonium sulphate precipitation, chromatography on DEAE‐Sephacel, Sephacryl S 300, S Sepharose and Q Sepharose. The native pyranose oxidase has a relative molecular mass (Mr) of 322800 ± 18300 as determined on the basis of its Stokes' radius (rs= 6.2 nm) and sedimentation coefficient (s20,w= 10.6), dynamic light‐scattering experiments, gradient‐gel electrophoresis and cross‐linking studies. SDS/PAGE resulted in one single polypeptide band of Mr76000 indicating that the enzyme consists of four subunits of identical size. The pyranose oxidase was shown to be an extremely stable glycoprotein with an isoelectric point of pH 5.3. It contains covalently bound FAD with an estimated stoichiometry of 3.6 molecules FAD/molecule enzyme. Pyranose oxidase was active with the substrates d‐glucose, d‐xylose, l‐sorbose, d‐galactose, methyl β‐d‐glucoside, maltose and d‐fucose. Regioselective oxidation of d‐glucose, l‐sorbose and d‐xylose to 2‐keto‐d‐glucose, 5‐keto‐d‐fructose and 2‐keto‐d‐xylose, was demonstrated by identifying the reaction products by mass spectroscopy 13C‐NMR spectroscopy and 1H‐NMR spectroscopy after purification and derivatization. The pH optimum of the pyranose oxidase was in the range pH 6.0–6.5 in 0.1 M potassium phosphate, and its activation energy ( H°) for the conversion of d‐glucose was 34.6 kJ/mol. The reactions with the sugars exhibited Michaelis‐Menten kinetics, and the Km values determined for d‐glucose, l‐sorbose, d‐xylose and oxygen were 1.1 mM, 50.0 mM, 29.4 mM and 0.65 mM, respectively. The activity of pyranose oxidase was only slightly affected by chelating reagents, thiol reagents, reducing reagents and bivalent cations each at 1 mM.
Hydrocortisone induces the synthesis of α2‐macroglobulin by rat mammary myoepithelial cells
Authors: WARBURTON Michael J. , COLES Brian , DUNDAS Sinclair R. , GUSTERSON Barry A. , O'HARE Michael J.
Published: March 2005

The effects of lactogenic hormones on protein secretion by 25.5‐G4.2.3 cells, a rat mammary myoepithelial cell line immortalised with a temperature‐sensitive T‐antigen, were investigated. Insulin, prolactin, estradiol and progesterone had no effect but hydrocortisone induced the secretion of two proteins with molecular masses of 175 kDa (p175) and 146 kDa (p146), 10–30‐fold and 5‐fold respectively. The induction of p175 and p146 synthesis by hydrocortisone was greater at 39.5°C than at 33°C reflecting the increased differentiation of 25.5–G4.2.3 cells at the higher temperature. Rat mammary epithelial cells did not synthesise p175. After addition of hydrocortisone to 25.5–G4.2.3 cells, there was a lag phase of 10 h before the synthesis of p175 was induced. Half‐maximal induction of p175 synthesis required a hydrocortisone concentration of 0.5 μM. p175 was identified as α2‐macroglobulin by N‐terminal amino‐acid sequence determination and immunoprecipitation with a specific antibody. Hydrocortisone induced a 5‐kb α2‐macroglobulin‐specific mRNA transcript in 25.5–G4.2.3 cells. Myoepithelial cells are responsible for synthesising the basement membrane around the rapidly expanding mammary alveoli during pregnancy. Myoepithelial cells also secrete metalloproteinases which are probably involved in turnover of the basement membrane. We suggest that increased levels of hydrocortisone during pregnancy induce the synthesis of α2‐macroglobulin, which is believed to be a potent inhibitor of metalloproteinases, by rat mammary myoepithelial cells to reduce proteolytic degradation of the basement membrane.
Purification and characterization of recombinant histidine‐tagged human platelet 12‐lipoxygenase expressed in a baculovirus/insect cell system
Authors: CHEN Xin‐Sheng , BRASH Alan R. , FUNK Colin D.
Published: March 2005

A baculoviral expression vector consisting of a sequence encoding a six‐histidine tag apposed to the human platelet 12‐lipoxygenase cDNA, under control of the polyhedrin promoter, was constructed. Recombinant 12‐lipoxygenase baculoviruses were used to infect Spodoptera frugiperda insect cells (Sf9). At 54 h post‐infection, maximal 12‐lipoxygenase activity and protein levels were achieved; the enzyme was purified to apparent homogeneity in a single step by nickel‐ion‐chelation chromatography in which the (His)6‐tagged 12‐lipoxygenase was eluted with 100 mM imidazole. The purified enzyme metabolized arachidonic acid almost exclusively to 12‐hydroperoxyeicosatetraenoic acid with little, if any, epoxyalcohol or reduction products and had a Vmax of 2–4 μmol min−1 mg protein−1, Km of 10 μM and kcat of ∼250 min−1. Linoleic acid, on the other hand, was converted to (13S)‐13‐hydroperoxy‐octadecadienoic acid at a rate which was about 2% of that obtained with arachidonic acid as substrate, but displayed the same Km. The enzyme was most active between pH 7.5–8 and activity was stimulated significantly in the presence of 0.006% Tween‐20. A polyclonal antibody to the recombinant enzyme was generated and found to recognize a single 75‐kDa band in platelets, human erythroleukemia cells and 12‐lipoxygenase baculoviral‐infected Sf9 cells by immunoblot and immunoprecipitation methods. 12‐Lipoxygenase protein represented 0.1% of the total soluble protein in platelet preparations. In immunofluorescence experiments 12‐lipoxygenase was observed in the cytoplasm of infected insect cells and in the human megakaryoblastic DAMI cell line. The isolation of large quantities of pure human platelet 12‐lipoxygenase should facilitate detailed biochemical structure/function studies.
Helical structure of basic proteins from spermatozoa
Authors: VERDAGUER Núria , PERELLÓ Margarita , PALAU Jaume , SUBIRANA Juan A.
Published: March 2005

We describe structural studies carried out with some basic proteins found in association with DNA in the spermatozoa of molluscs and echinoderms. We have studied proteins related to histone H1 as well as protamines. Structural prediction methods show that these proteins have a strong helical potential and contain several turns, mainly of the SPKK type. No β structures were found. Strong structural similarities have been detected between distantly related species. The presence of helical regions is confirmed by circular dichroism in trifluoroethanol solution. The influence of the SPKK turns is also evident in the CD spectra. In proteins which contain a high percentage of arginine we conclude that conventional prediction methods should be modified in order to allow for a higher helical potential for this amino acid residue. Synthetic peptides with a sequence present in the C‐terminal region of histone H1 have also been studied. It was found that octapeptides may only acquire a small amount of structure, whereas hexadecapeptides are 50–60% helical. These studies strongly suggest that both protamines and proteins related to the C‐terminal part of histone H1 interact with DNA mainly in the α‐helical conformation.
Purification and characterization of glyoxylate synthetase from greening potato‐tuber chloroplasts
Authors: JANAVE Machhindra T. , RAMASWAMY N. Krishnan , NAIR P. Madhusudanan
Published: March 2005

Glyoxylate synthetase catalyzing the condensation of two formate molecules into glyoxylate was purified to homogeneity by AcA‐34, Sepharose CL‐6B and DEAE‐Sepharose CL‐6B chromatography. A 150‐fold purification with a specific activity of 25 μmol · mg protein−1· 5 min−1 was obtained by this procedure. The reaction product was identified as glyoxylate. The enzyme was a tetramer having a molecular mass of 160 kDa with a subunit molecular mass of 40 kDa. The enzyme could be activated 3–4‐fold by the addition of 0.3 mM Fe2+ and 0.4 mM tetrahydrofolic acid to the reaction mixture. The requirement for Fe2+ and tetrahydrofolic acid was confirmed from the inhibition of enzyme by O‐phenanthroline and α‐aminopterin, respectively. The presence of a bound folate in the enzyme was indicated by the fluorescence emission at 450 nm and turbidity development in a Lactobacillus casei growth test. Fluorescence emission at 450 nm upon excitation at 280 nm indicated that the bound folate and the aromatic amino‐acid residues of the enzyme were in close vicinity. The enzyme was maximally active at 25°C and exhibited a pH optimum at 7.0. The concentration of substrate was optimal at 5.0 mM and Km for substrate was found to be 1.4 mM. Activation by Fe2+ did not alter the Km but caused an increase in Vmax. The enzyme contained about 14–16 disulfide linkages, of which two were found to be reduced by treatment with 2‐mercaptoethanol. The presence of excess 2‐mercaptoethanol in the enzyme was inhibitory, indicating that the two disulfide linkages reduced by 2‐mercaptoethanol were essential for activity. This was also confirmed by the inhibition of enzyme activity when reduced enzyme was treated with O‐phthalaldehyde, which formed a thioisoindole derivative with reduced thiol groups at the active site.
Energy‐transducing properties of primary proton pumps reconstituted into archaeal bipolar lipid vesicles
Authors: ELFERINK Marieke G. L. , DE WIT Janny G. , DRIESSEN Arnold J. M. , KONINGS Wil N.
Published: March 2005

Archaeal lipids differ considerably from eubacterial and eukaryotic lipids in their structure and physical properties. From the membranes of the extreme thermophilic archaea Sulfolobus acidocaldarius a tetraether lipid fraction was isolated, which can form closed and stable monolayer liposomes in aqueous media. The function of three different primary proton pumps originating from archaeal, bacterial and eukaryotic lipid sources have been studied after reconstitution in these liposomes: bacteriorhodopsin from the archaea Halobacterium halobium; cytochrome‐c oxidase from the thermophilic bacterium Bacillus stearothermophilus and cytochrome‐c oxidase from beef heart mitochondria. Liposomes composed of tetraether lipids form a competent matrix for all three exogenous proton pumps. Bacteriorhodopsin was inserted inside‐out in these liposomes, as normally observed in bilayer‐forming lipid. The activities of the two oxidases were inhibited at high tetraetherlipid concentration, probably due to the low fluidity of these membranes. Only bacteriorhodopsin, which originates from diether archaeal lipids is fully functional in the tetraether membranes.
Evidence that CTP:choline‐phosphate cytidylyltransferase is regulated at a pretranslational level in rat liver after partial hepatectomy
Authors: HOUWELING Martin , TIJBURG Lilian B. M. , VAARTJES Willem J. , BATENBURG Joseph J. , KALMAR Gabriel B. , CORNELL Rosemary B. , VAN GOLDE Lambert M. G.
Published: March 2005

Regulation of CTP:choline‐phosphate cytidylyltransferase activity was studied in regenerating rat liver. The formation of phosphatidylcholine from [14C]choline in hepatocytes isolated from regenerating liver at 22 h after surgery was increased 1.9‐fold when compared with hepatocytes from sham‐operated animals. This effect was accompanied by a 1.4‐fold increase in cytosolic cytidylyltransferase activity as well as by a 1.5‐fold increase in the amount of immunoreactive cytidylyltransferase protein, and a 1.7‐fold increase in [35S]methionine incorporation into cytidylyltransferase protein.

Northern blot analysis of cytidylyltransferase mRNA showed two signals at 1.5 and 5.0 kb. Partial hepatectomy caused a significant 2–3‐fold increase in the 1.5‐kb and 5.0‐kb messengers at 12 h after surgery. During the next 10 h after partial hepatectomy cytidylyltransferase mRNA levels slightly decreased, although they were still elevated in comparison with sham‐operated rats 20–22 h after surgery. In contrast to the elevated cytidylyltransferase mRNA levels, the amount of acetyl‐CoA carboxylase mRNA did not increase between 12 and 22 h after surgery, which is in line with the unchanged activity of this enzyme.

In conclusion, our data demonstrate that in regenerating liver phosphatidylcholine biosynthesis and cytidylyltransferase activity are regulated at a pretranslational level.
The bis(adenosin‐N6‐yl)alkanes, a family of potential dinucleoside‐polyphosphate analogue precursors
Authors: CHEN Haijuan , McLENNAN Alexander G.
Published: March 2005

A series of bis(adenosin‐N6‐yl)alkanes, in which two adenosine residues are linked via their N6 positions by alkyl bridges comprising between 2 and 14 methylene units, were synthesized as potential precursors to dinucleoside‐polyphosphate analogues. These compounds were moderately cytotoxic to mammalian cells, the toxicity increasing with the length of the alkyl chain. For example, the dose of bis(adenosin‐N6‐yl)dodecane, A[CH2]12A, leading to 50% inhibition of cell growth (ID50) for BHK fibroblasts, Walker 256 carcinoma cells and S‐49 T‐lymphoma cells were 90±8, 100±5 and 23±4 μM respectively. A significant amount of A[CH2]12A bound to serum albumin in the growth media; thus the ID50 for S‐49 cells grown in serum‐free medium was 9±2 μM. The corresponding bis‐cytidine analogues were much less toxic; however the presence of a second adenosine moeity/molecule had little significant effect on cell growth when compared to N6‐alkyladenosines. Toxicity to S‐49 cells was unaffected by the nucleoside‐transporter inhibitor nitrobenzylthioinosine and was even higher (ID50= 5 ± 0.5 μM) towards nucleoside‐transport‐deficient AE‐1 cells, showing that the analogues could pass freely through the plasma membrane. Interaction with A1 adenosine receptors was shown by displacement of [3H]N6‐R‐phenylisopropyladenosine (Kd= 6 nM) from rat adipocyte membranes, with Ki values of 45, 65, 85 and 390 nM for the compounds containing 12, 8, 6 and 4 methylene units, respectively. Affinity for human platelet membrane A2 adenosine receptors was about 100‐fold less, however the compounds were weak A2 agonists, producing up to a threefold increase in intracellular cyclic AMP in WI‐38/VA‐13 cells. Thus, these compounds behave, not surprisingly, as adenosine analogues. In addition, A[CH2]12A was metabolized in vitro and intracellularly by adenosine kinase (Ki= 70 nM) and adenylate kinase to yield a number of phosphorylated derivatives with the potential to act as diadenosine polyphosphate analogues. One of these, the bismonophosphate, was recognized by and inhibited adenylate kinase more effectively than adenosine(5′)tetraphospho(5′)adenosine (Ap4A, Ki= 3 μM).
The US‐1 element from the gene encoding rat poly (ADP‐ribose) polymerase binds the transcription factor Sp1
Authors: POTVIN Frédeéric , ROY René J. , POIRIER Guy G. , GUÉRIN Sylvain L.
Published: March 2005

By comparing the upstream DNA sequence of the rat and human genes encoding poly(ADP‐ribose) polymerase (PARP), we have defined a 16‐bp conserved region and designated it as US‐1 for ‘upstream sequence 1′. This element is homologous to the recently described binding site for the transcription factor Sp1 in the promoter sequence of the mouse p12 gene which encodes a protease inhibitor. Analyses in gel mobility shift assays revealed that a nuclear protein, produced by all tissue‐culture cells tested, specifically binds the US‐1 element. The pattern of shifted DNA · protein complexes obtained was strikingly similar to that for Sp1, which is supported by the positive displacement of these complexes by an oligomer containing the Sp1 binding site in gel shift competition experiments. Replacement of the Sp1 binding site from the basal promoter of the mouse p12 gene by the rPARP US‐1 element did not result in any significant variations in the level of expression of the chloramphenicol acetyltransferase (CAT) reporter gene upon transient transfection of tissue‐culture cells. However, when point mutations are introduced in the US‐1 element in a similar substitution experiment, a significant reduction in CAT gene expression could be observed. These data are consistent with Sp1 interacting with the US1 element. Results from DNase I footprinting experiments clearly indicated that purified Sp1 not only binds to the US‐1 element but also to four other closely located cis‐acting sites scattered in the promoter of the rat PARP gene, therefore suggesting that Sp1 is likely to modulate strongly the expression of that gene in different tissues.
Purification and characterization of guinea pig gastric phospholipase A2 of the pancreatic type
Authors: TOJO Hiromasa , YING Zhao , OKAMOTO Mitsuhiro
Published: March 2005

Guinea pig gastric mucosa and juice contained exceptionally high phospholipase‐A2 activity, whereas the activity in the pancreas was only minimal. Phospholipases A2 were purified to homogeneity from these three tissues. Structural evidence, including the sequence of the NH2‐terminal 41 residues, the amino‐acid composition and the molecular mass (13902 ± 3 Da) determined accurately by mass spectrometry, showed that the gastric mucosa enzyme belongs to the pancreatic type. An unique feature of the sequence is the substitution of Phe for the hitherto invariant Tyr28 in the calcium‐binding loop of pancreatic phospholipases A2. The affinity of the guinea pig enzyme for Ca2+ in the presence of substrate was, however, identical to that of the rat enzyme with Tyr28, suggesting the interaction of a phenolic hydroxyl group of the Tyr with its neighboring residues is not significantly linked to the binding of Ca2+. The NH2‐terminal sequences and immunochemical properties of the enzymes purified from the gastric juice and pancreas were identical to those of the gastric mucosa enzyme. The distribution of cells immunoreactive with anti‐(gastric PLA2) immunoglobulin in the stomach was quite similar to that of the chief cells. Unlike in pancreas of other animals, the prophospholipase A2 was not detectable in gastric mucosa or juice homogenates treated with diisopropyl fluorophosphate or in column effluents during purification under acidic conditions. An appreciable prophospholipase‐A2‐activating activity was not detectable in gastric mucosa extracts at low pH relevant to gastric juice, using rat prophospholipase A2 as substrate. This opposes the activation of secreted proenzyme in the gastric juice.
Human‐immunodeficiency‐virus transmembrane glycoprotein gp41 is an amino acceptor and donor substrate for transglutaminase in vitro
Authors: MARINEILLO Loredana , ESPOSITO Carla , DI PIERRO Prospero , COZZOLINO Anna , PUCCI Pietro , PORTA Raffaele
Published: March 2005

Recombinant gp41, the transmembrane glycoprotein of the human‐immunodeficiency‐virus (HIV) envelope, is an amino acceptor and donor substrate for transglutaminase in vitro. Gln51, Gln52, Gln66 and Lys77 residues were suggested as reactive sites, recognized by the enzyme, for possible cross‐linking reactions with gp120, CD4 or other receptor(s) occurring on the surface of HIV‐target cells. Soluble CD4, even though unable to function as an amino‐acceptor transglutaminase substrate, becomes active in the presence of gp41, negatively influencing the enzyme‐catalyzed incorporation of the polyamine spermidine into the transmembrane protein. These results suggest a possible role for transglutaminase in virus entry into host cells, via receptor‐mediated endocytosis, and/or in HIV‐induced CD4+ T‐cell depletion via apoptosis.
5‐Lipoxygenase‐activating protein stimulates the utilization of arachidonic acid by 5‐lipoxygenase
Authors: ABRAMOVITZ Mark , WONG Elizabeth , COX Martha E. , RICHARDSON Christopher D. , LI Chun , VICKERS Philip J.
Published: March 2005

5‐Lipoxygenase (5‐LO) and its activating protein (FLAP) are both required for cellular leukotriene (LT) synthesis, with 5‐LO catalyzing both the synthesis of (5S)‐5‐hydroperoxy 6,8,11,14‐eicosatetraenoic acid (5‐HPETE) from arachidonic acid and the subsequent synthesis of LTA4 from 5‐HPETE. We have previously expressed both human 5‐LO and human FLAP to high levels in Spodoptera frugiperda (Sf9) insect cells, using recombinant baculoviruses. To study the mechanism by which FLAP activates 5‐LO, we compared cellular 5‐LO activity in Sf9 cells expressing this enzyme to that in Sf9 cells coexpressing FLAP and 5‐LO. In this system, FLAP stimulates the utilization of arachidonic acid by 5‐LO as a substrate, and increases the efficiency with which 5‐LO converts 5‐HPETE to LTA4. LT synthesis in cells coexpressing FLAP and 5‐LO is inhibited by 3‐[1‐p‐chlorophenyl)‐5‐isopropyl‐3‐tert‐butylthio‐1H‐indol‐2‐yl]‐2,2‐dimethyl‐propanoic acid (MK‐886), an LT biosynthesis inhibitor which specifically binds to FLAP. These studies in Sf9 cells, together with our recent demonstration that FLAP specifically binds arachidonic acid, suggests that FLAP activates 5‐LO by acting as an arachidonic acid transfer protein.
Cell‐cell adhesion by homophilic interaction of the neuronal recognition molecule axonin‐1
Authors: RADER Christoph , STOECKLI Esther T. , ZIEGLER Urs , OSTERWALDER Thomas , KUNZ Beat , SONDEREGGER Peter
Published: March 2005

The axonal surface glycoprotein axonin‐1, which occurs both as a glycosyl‐phosphatidylinositol‐anchored membrane‐bound form and a secreted form, promotes neurite outgrowth and is thought to be involved in axon‐guidance mechanisms in the developing nervous system. Recently, we have demonstrated that the neurite‐outgrowth‐promoting activity of axonin‐1, presented as substratum for cultured neurons, is mediated by a heterophilic interaction with the axonal glycoprotein neuronglia cell‐adhesion molecule (Ng‐CAM). Here we present evidence for homophilic (like‐like) binding among axonin‐1 molecules. Axonin‐1 was heterologously expressed in myeloma cells. Clonal cell lines, with exposed membrane‐bound axonin‐1 at their surface, formed large multicellular aggregates. Incubations of transfected and parental myeloma cells, under a series of different conditions, revealed homophilic axonin‐1/axonin‐1 interactions across the intermembrane space as the molecular mechanism promoting stable cell‐cell contacts. Using structural and functional characterisation, recombinant axonin‐1 was very similar to native axonin‐1, suggesting that homophilic axonin‐1 interactions are also established in neurons. The capability of axonin‐1 to interact with both Ng‐CAM and other axonin‐1 molecules might contribute to the formation of macromolecular networks at contact sites of growth cones and axons, comprising molecules of both membranes, and thus represent a mechanism for regulating neurite outgrowth and pathfinding.
Interaction of type IV collagen with the isolated integrins α1β1 and α2β1
Authors: KERN Andreas , EBLE Johannes , GOLBIK Ralph , KÜHN Klaus
Published: March 2005

The triple‐helical cyanogen‐bromide‐derived fragment CB3[IV] of collagen IV, located 100 nm from the N‐terminus of the molecule, contains the binding sites for the integrins α1β1 and α2β1. To investigate the interaction of these integrins and collagen IV, we performed solid‐phase and inhibition assays using as receptor isolated α1β1 and α2β1. The ligands used were the binding‐site‐bearing trimeric peptide CB3[IV] and its shorter tryptic fragments F1–F4. Using titration curves, in which the binding of soluble receptors to coated ligands and the binding of soluble ligands to coated receptors were analyzed, the binding sites for α1β1 and α2β1 were in different but adjacent areas of CB3[IV]. Triple‐helical conformation and distinct primary structures were required for the interaction. Dissociation constants (Ka), for the affinity of integrins for collagen IV, were determined in the 1‐nM range in the presence of Mn2+ and Mg2+. In the absence of Mn2+, the Kd values indicated a 30–60‐fold decrease in the affinities, which for α2β1 was further reduced by adding Ca2+. In the presence of Ca2+ and Mg2+ the affinity of collagen IV for α1β1 was four‐times higher than for α2β1.
Synthesis of 3‐arsonoalanine and its action on aspartate aminotransferase and aspartate ammonia‐lyase
Authors: ALI Bassam R. S. , DIXON Henry B. F.
Published: March 2005

dl‐3‐Arsonoalanine has been synthesized by the Strecker synthesis from the unstable compound arsonoacetaldehyde. It inactivates pig heart cytosolic aspartate aminotransferase and inhibits aspartate ammonia‐lyase by competing with aspartate (Ki/Km 0.23). The fumarate analogue (E)‐3‐arsonoacrylic acid and the malate analogue (RS)‐3‐arsonolactate also inhibit fumarate hydratase, competing with fumarate (Ki/Km 1.8) and malate (Ki/Km 1.6) respectively. Attempted non‐enzymic transamination of 3‐arsonoalanine gave elimination of arsenite, in contrast with the transamination of 3‐phosphonoalanine, which is either successful or leads to loss of phosphate.
Increase of specificity of RNase from Bacillus amyloliquefaciens (barnase) by substitution of Glu for Ser57 using site‐directed mutagenesis
Authors: YAKOVLEV Gennady I. , MOISEYEV Gennady P. , STRUMINSKAYA Nina K. , ROMAKHINA Elena R. , LESHCHINSKAYA Inna B. , HARTLEY Robert W.
Published: March 2005

Bacterial ribonucleases from Bacillus amyloliquefaciens and Bacillus intermedius show the specificity towards the nature of a nucleoside at the O3′ end of the phoshodiester bond to be split in the preference order G> A≫ U>C in the cleavage reactions of polynucleotides. It follows from the X‐ray data that the substrate guanosine base is bound at the active site of these RNases in the same manner as for high‐specificity guanylic RNases. We supposed that the difference in specificity for the two types of RNases is due to the additional hydrogen bond between the protein and a purine base in the case of bacterial guanyl‐preferring RNases in contrast to the high‐specificity guanylic RNases. To examine this hypothesis we prepared the Ser57→Glu mutant of B. amyloliquefaciens, in which this hydrogen bond is eliminated. Kinetic studies demonstrate that the specificity of this mutant towards guanylic substrates is 35‐times greater than that of the wild‐type RNases from B. amyloliquefaciens and close to that of RNases T1.
Structural study of the oligosaccharide moieties of sphingolipid activator proteins, saposins A, C and D obtained from the spleen of a Gaucher patient
Authors: ITO Keita , TAKAHASHI Noriko , TAKAHASHI Akira , SHIMADA Ichio , ARATA Yoji , O'BRIEN John S. , KISHIMOTO Yasuo
Published: March 2005

We have determined and compared the structures of the oligosaccharide moieties of saposin A, C and D purified from the spleen of a patient with Gaucher disease. These saposins, together with saposin B, are small glycoproteins, derived from separate domains of a single precursor, prosaposin, and are required for the lysosomal hydrolysis of various sphingolipids. The characteristic features of the oligosaccharide moieties of saposin A are (a) the predominance of a fucosylated trimannosyl core structure and (b) the occurrence of several different oligomannose‐type and N‐acetyllactosamine‐type oligosaccharides. Saposin C contains (a) a predominance of oligomannose‐type oligosaccharides and monoantennary oligosaccharides and (b) the presence of four different oligosaccharides having bisecting N‐acetylglucosamine residues (found only in this saposin). Saposin D is distinguished by the occurrence of oligomannose‐type oligosaccharides, which comprise nearly 90% of its total oligosaccharides. The possible reasons for the unique glycosylation of each saposin is discussed.
Rapid dephosphorylation of microtubule‐associated protein 2 in the rat brain hippocampus after pentylenetetrazole‐induced seizures
Authors: DÍEZ‐GUERRA F. Javier , AVILA Jesús
Published: March 2005

We have studied the effect of Pentylenetetrazole (PTZ)‐induced seizures on the state of phosphorylation of microtubule‐associated protein 2 (MAP‐2) from rat hippocampus. A method for the in vivo32P‐labeling of hippocampal proteins has been established, consisting of intracerebro‐ventricular injection of 32PO4 of high specific activity. The results obtained indicate that PTZ induces a rapid and transient dephosphorylation of high‐molecular‐mass MAP‐2, which is prevented when the N‐methyl‐d‐asparatate receptor antagonist MK‐801 is previously administered. Phosphopeptide mapping of 32P‐labeled MAP‐2 obtained from hippocampi of PTZ‐treated rats reveals a pattern of phosphorylation distinct from that obtained from control saline‐treated rats or MK‐801 plus PTZ treated rats. We discuss the possible implications of N‐methyl‐d‐aspartate‐receptor activation and MAP‐2 dephosphorylation on the plastic changes induced in rat brain hippocampus after induced epileptiform activity.
Strategies of antioxidant defense
Authors: SIES Helmut
Published: March 2005

Cellular protection against the deleterious effects of reactive oxidants generated in aerobic metabolism, called oxidative stress, is organized at multiple levels. Defense strategies include three levels of protection; prevention, interception, and repair. Regulation of the antioxidant capacity includes the maintenance of adequate levels of antioxidant and the localization of antioxidant compounds and enzymes. Short‐term and long‐term adaptation and cell specialisation in these functions are new areas of interest. Control over the activity of prooxidant enzymes, such as NADPH oxidase and NO synthases, is crucial. Synthetic antioxidants mimic biological strategies.
Primary structure of the human muscle acetylcholine receptor
Authors: BEESON David , BRYDSON Martin , BETTY Maria , JEREMIAH Steve , POVEY Sue , VINCENT Angela , NEWSOM‐DAVIS John
Published: March 2005

cDNA sequences encompassing the full coding region for the human muscle acetylcholine receptor (AChR) ɛ and γ subunits have been isolated. The deduced amino‐acid sequences indicate that the mature ɛ subunit contains 473 amino acids and is preceded by a 20‐amino‐acid signal peptide. As predicted from genomic clones, the γ subunit contains 495 amino acids preceded by a 22‐amino‐acid signal peptide. In common with the human α, β, γ, and δ subunits the ɛ subunit is highly conserved between mammalian species. The ɛ subunit gene is not closely linked to the γ and δ subunits on chromosome 2 but rather is located with the β subunit on chromosome 17. Expression of the α‐, β‐, γ‐, δ‐, and ɛ‐subunit cRNAs in rabbit‐reticulocyte lysates followed by analysis on SDS/PAGE show glycosylated proteins with apparent molecular masses of 44–60 kDa.
1H‐NMR analysis of turkey egg‐white lysozyme and comparison with hen egg‐white lysozyme
Authors: BARTIK Kristin , DOBSON Christopher M. , REDFIELD Christina
Published: March 2005

The complete main chain and approximately 75% of the side chain 1H‐NMR assignments of the 129‐residue protein, turkey egg‐white lysozyme, are presented. NOE data, hydrogen‐exchange rates, chemical shifts and coupling constants are reported and are indicative of a structure in solution that is essentially identical to that of the homologous hen egg‐white lysozyme. The NH‐αCH coupling constants of turkey lysozyme are compared to torsion‐angle data from three crystal structures of the protein and the results are interpreted in terms of crystal‐structure resolution and refinement.
Dimeric forms of cholinesterase in Sipunculus nudus
Authors: TALESA Vincenzo , PRINCIPATO Giovanni B. , GIOVANNINI Elvio , DI GIOVANNI M. Vittoria , ROSI Gabriella
Published: March 2005

In developing a research on the cholinesterase (ChE) evolution in Invertebrata, this enzyme was studied in the unsegmented marine worm Sipunculus nudus. ChE activity was solubilized through three successive steps of extraction. These fractions are noted as low‐salt (LSS), detergent (DS) and high‐salt soluble (HSS) and represent 27%, 68% and 5% of total activity, respectively. LSS and DS ChE were purified to homogeneity by affinity chromatography on edrophonium‐Sepharose gel. Purification factors of 1700 (LSS) and 1090 (DS) were obtained. The small amount of HSS ChE prevented a similar purification and an extensive characterization. Based on SDS/PAGE and densitygradient centrifugation, both LSS and DS enzymes show a Mr value of about 130000 and are likely G2 globular dimers of a 67000 subunit. Moreover, LSS ChE seems to be an amphiphilic form including a hydrophobic domain, while DS ChE is probably linked to the cell membrane by a phosphatidylinositol anchor. Both LSS and DS enzymes hydrolyze at the highest rate propionylthiocholine. However, they also show a fairly high catalytic efficiency with other thiocholine esters as substrates, thus suggesting a wide and little‐specialized conformation of the active site. Based on immunological cross‐reactivity trials, LSS and DS ChE from S. nudus show a reduced structural affinity with a molluscan (Murex brandaris) enzyme. HSS ChE, an acetylcholinesterase, is also solubilized by heparin, like typical vertebrate HSS asymmetric enzymes. However, it lacks fastsedimenting forms and an enzyme‐anchoring collagenous structure.
Regulation of malic‐enzymes‐gene expression by cAMP and retinoic acid in differentiating brown adipocytes
Authors: HERNANDEZ Arturo , GARCIA‐JIMENEZ Custodia , SANTISTEBAN Pilar , OBREGON María J.
Published: March 2005

Brown adipose tissue (BAT) is composed of highly specialized cells, whose main function is to produce heat under adrenergic stimulation, uncoupling oxidative phosphorylation. For this function, lipogenesis must be accurately regulated. Malic enzyme has a central role in lipogenesis and is strongly expressed in brown adipocytes. In this work, we study the modulation by adrenergic stimuli, cAMP effectors and retinoic acid on the induction produced by insulin and 3,5,3′‐triiodothyronine on malic‐enzyme‐gene expression. Primary cultures of differentiating brown adipocytes have been used. The results obtained demonstrate that physiological doses of norepinephrine do not modify malic‐enzyme mRNA levels when acting alone, but considerably reduce the induction produced by insulin, 3,5,3′‐triiodothyronine or both together. Other cAMP inducers such as glucagon, forskolin or 8‐bromo‐cAMP, greatly inhibit both, basal and 3,5,3′‐triiodothyronine‐induced malic‐enzyme‐gene expression. Retinoic acid abolishes basal and also inhibits 3,5,3′‐triiodothyronine‐induced malic‐enzyme‐gene expression.
Ribosomes terminated in vitro are in a tight association with non‐phosphorylated elongation factor 2 (eEF‐2) and GDP
Authors: DAVYDOVA Elena K. , MALININ Nikolai L. , OVCHINNIKOV Lev P.
Published: March 2005

A proportion of the ribosome population in the eukaryotic cell is present in the form of single 80‐S ribosomes. These are not involved in translation and are tighly associated with eukaryotic elongation factor 2 (eEF‐2). The factor dissociates from ribosomes when it is ADP‐ribosylated. Attempts at reconstitution of such complexes from ribosomal subunits and eEF‐2 were not successful.

We have shown that monomeric ribosomes in a tight complex with eEF‐2 can be obtained in vitro as terminated ribosomes in a reconstituted translation system containing isolated polyribosomes, elongation factors and pH5 enzymes (all from rabbit reticulocytes). Incubation of the system with radioactive GTP demonstrated that terminated ribosomes contain GDP. ADP‐ribosylation of eEF‐2 bound to terminated ribosomes by diphtheria toxin leads to dissociation of both eEF‐2 and GDP to the same extent. Thus the presence of GDP in terminated ribosomes is eEF‐2 dependent. Ribosomes terminated in vitro as well as native single ribosomes contain the non‐phosphorylated form of eEF‐2.

We assume that tight association of terminated ribosomes with the non‐phosphorylated form of eEF‐2 excludes both the ribosomes and active eEF‐2 from the translational cycle and thus, maintains the optimal proportion of translating ribosomes and free eEF‐2 in the cell.
Stabilized NMR structure of human parathyroid hormone(1–34)
Authors: BARDEN Julian A. , CUTHBERTSON Ruth M.
Published: March 2005

The structure of the biologically‐active N‐terminal region of human parathyroid hormone, PTH(1–34), was investigated in the presence of 10% trifluoroethanol using two‐dimensional proton NMR spectroscopy, distance geometry and dynamic simulated annealing. Complete assignments of all backbone and side chain hydrogens were made with the aid of totally correlated spectroscopy (TOCSY) experiments, providing through‐bond 1H‐1H connectivities, and NOESY providing through‐space and sequential backbone connectivities. Distance and angle constraints were used in the distance geometry algorithm DIANA II to generate a family of structures satisfying all inputs. The lowest energy structures were further refined using the dynamic‐simulated‐annealing protocol XPLOR 3. The major structural features evident in 10% trifluoroethanol are two segments of α‐helix extending from residues Glu4 to Lys14/His14 and Ser17 to Asp30. A short length of unordered structure joined the two spans of helix. The structures of the N‐terminal regions (4–13/14) agreed closely with the structure found in human parathyroid‐hormone‐related protein (PTHrP)(1–34) obtained earlier by Ray, Barden and Kemp [19]. However, PTHrP(16–19) exhibited a reverse turn which is incorporated in an extended helix in PTH. The consequent interactions between several hydrophobic residues in the C‐terminal helix is present as a more standard loop of helix in PTH. Comparisons between the structures of the two hormones has enabled the probable location and structure of the PTH. Comparisons between the structures of the two hormones has enabled the probable location and stuctufre of the PTH receptor‐binding site to be placed in the segment of amphiphilic α‐helix (24–31).
Extensive accumulation of an extracellular l‐amino‐acid oxidase during gametogenesis of Chlamydomonas reinhardtii
Authors: VALLON Olivier , BULTÉ Laurence , KURAS Richard , OLIVE Jacqueline , WOLLMAN Françis‐André
Published: March 2005

In a previous study [Bulté, L. & Wollman, F.‐A. (1992) Eur. J. Biochem. 204, 327–336], we identified a novel gamete‐specific polypeptide of Chlamydomonas reinhardtii, Mα. This 66‐kDa polypeptide reacts with antibodies to cytochrome f and accumulates in gametes only in conditions that promote destabilisation of the cytochrome b6lf complex. Here, we show that Mα is not a modification product of cytochrome f, but is part of protein M, a high‐molecular‐mass l‐amino‐acid oxidase located in the periplasm. It catalyzes oxidation of all l‐amino acids tested, except cysteine. Using phenylalanine as a substrate, saturation of the enzymatic rate is reached at 2 μM. These characteristics suggest that protein M may operate in vivo as an efficient scavanger of ammonium from extracellular amino acids. The enzyme contains non‐covalently bound FAD. It exists in two forms with essentially similar enzymatic properties, of 1.2–1.3 MDa and 0.9–1.0 MDa, respectively. The lighter form is an oligomer of Mα, while the heavier form contains, in addition to Mα, a second polypeptide of 135 kDa, Mβ, in a molar ratio of 3–4 Mα/Mβ. Both polypeptides are glycosylated.
Primary structure of human milk octa‐, dodeca‐ and tridecasaccharides determined by a combination of 1H‐NMR spectroscopy and fast‐atom‐bombardment mass spectrometry
Authors: HAEUW‐FIEVRE Sophie , WIERUSZESKI Jean‐Michel , PLANCKE Yves , MICHALSKI Jean‐Claude , MONTREUIL Jean , STRECKER Gérard
Published: March 2005

Two octasaccharides, two dodecasaccharides and a tridecasacharide have been isolated from human milk by a combination of paper chromatography, high‐performance liquid chromatography and high‐performance anion‐exchange chromatography. Their structural analysis investigated by 400‐MHz 1H‐ and 13C‐NMR spectroscopy and by mass spectrometry led to the following structures.

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The two octasaccharides have been previously characterized in human milk; the complete assignment of the 1H and 13C spectra is reported here. One of the dodecasaccharides is a tetrafucosyl derivative with a new core: typI‐(β1–3)‐typeII‐(β1–3)‐typeII‐(β1–3)‐Gal(β1–4)Glc, where typeI = Gal(β1–3)GlcNAc and typeII = Gal(β1–4)GlcNAc. These oligosaccharides display simultaneously Lewisb and Lewisx determinants or Lewisa, Lewisb and Lewisx determinants.