bluuebunny/crossref_metadata_2025 huggingface dataset:
Guanosine triphosphate catabolism in purine nucleoside phosphorylase deficient human B lymphoblastoid cells
Authors: Barankiewicz J. , Stein L.D. , Cohen A.
Published: July 2002

GTP catabolism induced by sodium azide or deoxyglucose was studied in purine nucleoside phosphorylase (PNP) deficient human B lymphoblastoid cells. In PNP deficient cells, as in control cells, guanylate was both dephosphorylated and deaminated but dephosphorylation was the major pathway. Only nucleosides were excreted during GTP catabolism by PNP deficient cells and the main product was guanosine. The level of nucleoside excretion was largely affected by intracellular orthophosphate (Pi) level. In contrast, normal cells excreted nucleosides only at low Pi level while at high Pi levels, purine bases (guanine and hypoxanthine) were exclusively excreted. PNP deficiency had no effect on the extent of GMP deamination.
H1 histone, polylysine and spermine facilitate nucleosome assembly in vitro
Authors: Bogdanova E.S.
Published: July 2002

Nucleosome formation has been studied in a system containing relaxed Col El DNA, core histones and an extract of Drosophila embryos. The formation of nucleosomes was established by the introduction of supercoils into DNA. The degree of DNA supercoiling was shown to be higher if nucleosomes were assem‐bled in the presence of the H1 histone, polylysine (M
r 20000) or spermine. These agents do not stimulate relaxation and are the more effective the earlier they are added to the reaction. Thus, the H1 histone, polylysine and spermine facilitate nucleosome assembly in vitro.
Sequence conservation in the N‐terminal domain of histone H1
Authors: Böhm L. , Mitchell T.C.
Published: July 2002

Histone H1
N‐domain
Conserved segment
Citrate synthase: an immunochemical investigation of interspecies diversity
Authors: Pullen Ann M. , Budgen Nigel , Danson Michael J. , Hough David W.
Published: July 2002

Citrate synthase
Immunochemistry
Enzyme diversity
O2−dependent lipid peroxidation does not affect the molecular order in hepatoma microsomes
Authors: Masotti L. , Cavatorta P. , Ferrari M.B. , Casali E. , Arcioni A. , Zannoni C. , Borrello S. , Minotti G. , Galeotti T.
Published: July 2002

Microsomal membranes from rat liver and from the fast‐growing Morris hepatoma 3942A have been peroxidized to different extents and the order parameter of the membranes measured by fluorescence depolarization of the probe 1,6‐diphenyl‐1,3,5‐hexatriene. The data have been analysed by applying a mathematical approach that takes into account simultaneously static and dynamic fluorescence parameters. It appears that tumour membranes are more ordered than the control and their order parameter does not increase with greater exposure to the action of O−
2 radicals in contrast to liver membranes. The fatty acid composition of the membrane lipids has been studied under different experimental conditions and correlated to the behaviour of the physical parameter.
Inactivation of basal glycogen synthase by glucagon and epinephrine in hepatocytes from fed rats
Authors: Bosch Fàtima , Ciudad Carles J. , Guinovart Joan J.
Published: July 2002

Glucagon and epinephrine promote the inactivation of basal glycogen synthase in hepatocytes isolated from fed rats. However, this effect is only observable when the activation state of glycogen synthase is measured using the low glucose‐6‐P/high glucose‐6‐P activity ratio assay. This inactivation is the consequence of an increase in the kinetic parameters (S
0.5 for UDP‐glucose and M
0.5 for glucose‐6‐P) of the enzyme. Therefore, this work demonstrates these hormones are also able to control glycogen synthase from fed animals.
Lipoprotein‐binding proteins in the human platelet plasma membrane
Authors: Koller Elisabeth
Published: July 2002

The binding of homologous plasma lipoproteins to specific receptor proteins in the plasma membrane of human blood platelets was studied by ligand blotting techniques. HDL3, HDL2 and LDL showed saturable binding to three bands of 156, 130 and 115 kDa, respectively. This binding was not markedly affected by the presence or absence of Ca2+ nor by covalent modification of lysine and arginine residues of the apoprotein moieties. However, it can be almost completely reversed by the addition of heparin or suramin.
Solubilization and partial purification of UDP‐galactose:diacylglycerol galactosyltransferase activity from spinach chloroplast envelope
Authors: Covès Jacques , Block Maryse A. , Joyard Jacques , Douce Roland
Published: July 2002

We have developed procedures to solubilize the envelope UDP‐galactose:diacylglycerol galactosyltransferase activity and to assay this enzyme after solubilization with a zwitterionic non‐denaturing detergent (CHAPS) and fractionation. From solubilized envelope membranes isolated from intact spinach chloroplasts, we were able to prepare by chromatography on hydroxyapatite a fraction enriched (6–7‐fold) in this enzyme responsible for monogalactosyldiacylglycerol synthesis.
8‐Bromoguanosine 3′:5′‐cyclic monophosphate decreases intracellular free calcium concentrations in cultured vascular smooth muscle cells from rat aorta
Authors: Kai Hisashi , Kanaide Hideo , Matsumoto Takahiro , Nakamura Motoomi
Published: July 2002

The effects of 8‐bromoguanosine 3′:5′‐cyclic monophosphate (8‐Br cGMP) on intracellular free calcium concentrations ([Ca2+]i) in cultured rat aortic vascular smooth muscle cells (VSMCs) loaded with fura‐2 were recorded microfluorometrically. Irrespective of whether VSMCs were at rest (in 5 mM K+ PSS), under Ca2+ depletion (in Ca2+‐free medium for 10 min) and K+ depolarization (in high K+ PSS), [Ca2+]i was actively reduced and reached a new and lower steady‐state level with the application of 8‐Br cGMP. This may be the first and direct evidence that cGMP, a putative mediator of various vasodilators, actively reduces [Ca2+]i in VSMCs.
One‐electron reduction of an anthracycline antibiotic carminomycin by a human erythrocyte redox chain
Authors: Peskin Alexander V. , Bartosz Grzegorz
Published: July 2002

Human erythrocyte membranes catalyse the NAD(P)H‐dependent generation of the semiquinone of an adriamycin‐type antibiotic carminomycin under anaerobic conditions. The maximal yield of the antibiotic radical is about 4‐fold higher in the presence of NADPH than of NADH. The possible significance of the antibiotic reduction to the semiquinone by a human erythrocyte membrane redox chain for the clinical usage of these antibiotics is discussed.
N‐(2‐Hydroxyethyl)phenazinium derivatives of oligonucleotides as effectors of the sequence‐specific modification of nucleic acids with reactive oligonucleotide derivatives
Authors: Kutyavin I.V. , Podyminogin M.A. , Bazhina Yu.N. , Fedorova O.S. , Knorre D.G. , Levina A.S. , Mamayev S.V. , Zarytova V.F.
Published: July 2002

It has been found that mono‐ and especially diphenazinium derivatives of oligonucleotides complementary to the DNA sequence adjacent to the target sequence of the addressed alkylation of DNA, significantly enhance the extent and specificity of alkylation with p‐(N‐2‐chloroethyl‐N‐methylamino)benzylamide derivatives of the addressing oligonucleotides, thus playing the role of effector of the sequence‐specific (complementary addressed) modification.
400‐MHz 1H‐NMR spectroscopy of fucosylated tetrasialyl oligosaccharides isolated from normal and cirrhotic α1‐acid glycoprotein
Authors: Wieruszeski J.M. , Fournet B. , Konan D. , Biou D. , Durand G.
Published: July 2002

The comparative study of fucosylated tetrasialyl‐oligosaccharides isolated from normal and cirrhotic α1‐AGP was performed using permethylation and 400‐MHz 1H‐NMR spectroscopy. These results clearly show the tetraantennary structure of these two oligosaccharides with hyperfucosylation for the tetrasialylated fraction from cirrhotic α1‐AGP. In the latter oligosaccharide the simultaneous presence on two antennae (7 and 7′) of the sialosyl Lewis X determinant NeuAc‐(α2–3) Gal(β1–4) [Fuc(αl–3)l GlcNAc has been observed. Moreover the 5 and 5′ antennae were α2–6 sialylated but without fucose.
Redox loops and proton pumps
Authors: Malmström Bo G.
Published: July 2002

In a Review‐Hypothesis, Mitchell [(1987) FEBS Lett. 222, 235–245] has recently suggested possible molecular mechanisms for proton translocation by cytochrome oxidase. In describing these mechanisms, he extended his own concept of a redox loop in a manner expected to lead to confusion. He also stated that the term redox‐linked proton pump implies an indirect coupling between electron transfer and proton translocation, and that this type of coupling is very difficult to test experimentally. Here it is argued that the original meaning of a redox loop should be maintained, and proper definitions of the terms redox‐linked proton pump and direct or indirect coupling are formulated. In addition, it is reasoned that both types of coupling are amenable to experimental tests.
Molecular cloning and amino acid sequence of human enkephalinase (neutral endopeptidase)
Authors: Malfroy Bernard , Kuang Wun-Jing , Seeburg Peter H. , Mason Anthony J. , Schofield Peter R.
Published: July 2002

We have isolated a cDNA clone encoding human enkephalinase (neutral endopeptidase, EC 3.4.24.11) in a λgt10 library from human placenta, and present the complete 742 amino acid sequence of human enkephalinase. The human enzyme displays a high homology with rat and rabbit enkephalinase. Like the rat and rabbit enzyme, human enkephalinase contains a single N‐terminal transmembrane region and is likely to be inserted through cell membranes with the majority of protein, including its carboxy‐terminus, located extracellularly.
Identification of an active‐site residue in subunit S1 of pertussis toxin by photocrosslinking to NAD
Authors: Cockle Stephen A.
Published: July 2002

The site of interaction of NAD with the isolated S1 subunit of pertussis toxin was investigated by photoaffinity labelling. When S1 was irradiated at 254 nm in the presence of [carbonyl‐14C]‐ or [adenine‐14C]NAD, the uptake of radioactivity was equivalent to 0.75 and 0.1 mol/mol respectively, while the NAD glycohydrolase activity was abolished. Inactivation was thus accompanied by crosslinking of the nicotinamide portion of NAD to the protein. Sequence determination of purified radioactive peptides indicated that Glu‐129 was a major site of labelling. This residue is therefore closely associated with either NAD binding or hydrolysis.
Fatty acid acylated antibodies against virus suppress its reproduction in cells
Authors: Kabanov A.V. , Ovcharenko A.V. , Melik-Hubarov N.S. , Bannikov A.I. , Alakhov V.Yu. , Kiselev V.I. , Sveshnikov P.G. , Kiselev O.I. , Levashov A.V. , Severin E.S.
Published: July 2002

A method for suppression of virus reproduction in cells using fatty acylated antiviral antibodies, which in contrast to non‐modified antibodies are capable of intracellular penetration, has been suggested. The addition of stearoylated antiviral antibodies to influenza A/Chili virus‐infected cells causes a 100‐fold suppression of virus reproduction. Non‐modified antibodies do not produce any effect on virus reproduction.
Stem bromelain: Amino acid sequence and implications for weak binding of cystatin
Authors: Ritonja Anka , Rowan Andrew D. , Buttle David J. , Rawlings Neil D. , Turk Vito , Barrett Alan J.
Published: July 2002

The amino acid sequence of stem bromelain, the major cysteine proteinase from pineapple stem is described. It shows that the enzyme is a member of the papain superfamily of cysteine proteinases, but is not very closely related to any other known member of this group. The sequence shows mutation or deletion of several residues that have been conserved in cysteine proteinases examined previously, including Asn‐175 (papain). We suggest that some of these changes have the effect of altering the active‐site geometry of stem bromelain, and that this accounts for the resistance of the enzyme to inhibition by cystatins and E‐64 [L‐3‐carboxy‐2,3‐trans‐epoxypropionylleucylamido(4‐guanidino)butane].
Inhibition of lipoprotein lipase by the receptor‐binding domain of apolipoprotein E
Authors: McConathy Walter.J. , Wang Chi-Sun
Published: July 2002

A synthetic peptide (residues 139–153) corresponding to the receptor‐binding domain of apolipoprotein E (ApoE) was tested for lipoprotein lipase (LPL) inhibitory properties. In systems using both natural and synthetic substrates, inhibition of LPL was observed. Using the synthetic substrate, 50% inhibition was observed at 50 μM while high concentrations completely inhibited LPL activity. These studies suggest an additional functional role for the receptor‐binding domain of ApoE — modulation of LPL activity.
Reconstitution of electron transport in photosystem I with PsaC and PsaD proteins expressed in Escherichia coli
Authors: Zhao Jindong , Warren Patrick V. , Li Ning , Bryant Donald A. , Golbeck John H.
Published: July 2002

A fusion protein, denoted PsaCl, which contains an amino‐terminal extension of five amino acids (MEHSM...) and is derived from an in vitro modified form of the psaC gene of Synechococcus sp. PCC 7002, has been over‐expressed in Escherichia coli. The product of the psaD gene of Nostoc sp. PCC 8009 has similarly been over‐expressed. The PsaCl and PsaD proteins can be combined with the photosystem I core protein of Synechococcus sp. PCC 6301 to reconstitute electron transport from P700 to the terminal FA/FB acceptors. Reconstitution was found to be absolutely dependent on reinsertion of the iron‐sulfur clusters in the PsaCI apoprotein and on the presence of the PsaD protein. This implies that the PsaCl holoprotein does not bind solely to the PsaA/PsaB heterodimer but rather that its interaction with these proteins is mediated through the PsaD protein.
Eukaryotic DNA methylation: facts and problems
Authors: Doerfler Walter , Toth Miklos , Kochanek Stefan , Achten Sabine , Freisem-Rabien Uta , Behn-Krappa Annett , Orend Gertraud
Published: July 2002

Patterns of DNA methylation in complex genomes like those of mammalian cells have been viewed as indicators of different levels of genetic activities. It is as yet unknown how these complicated patterns are generated and maintained during cell replication. There is evidence from many different biological systems that the sequence‐specific methylation of promoters in higher eukaryotes is one of the important factors in controlling gene activity at a long‐term level. In general, the fifth nucleotide 5‐methyldeoxycytidine can be considered as a modulator of protein‐DNA interactions. The degree and direction of this modulation has to be assessed experimentally in each individual instance. The establishment of de novo patterns of DNA methylation is characterized by the gradual non‐random spreading of DNA methylation by an essentially unknown mechanism. In this review, some of the general concepts of DNA methylation in mammalian systems are presented, and research currently performed in the authors' laboratory has been summarized.
Characterization of a chicken polyubiquitin gene preferentially expressed during spermatogenesis
Authors: Mezquita Jovita , Mezquita Cristóbal
Published: July 2002

We have previously reported that a chicken polyubiquitin gene (Ub II) not expressed under normal or heat shock conditions in chick fibroblasis is transcribed during spermatogenesis [(1987) Nucleic Acids Res. 15, 9604]. The level of Ub II mRNA is several‐fold higher in testis cells than in somatic tissues. The gene Ub II possesses characteristic features not seen in the polyubiquitin gene expressed in heat shock conditions (Ub I). The 5′ noncoding region of Ub II shows the consensus cAMP regulatory element (CRE) followed immediately downstream by a CA dinucleotide. It has been proposed that this extended CRE may be involved in the coordinate expression of various genes during spermatogenesis.
Cleavage of the precursor of pea chloroplast cytochrome ƒ by leader peptidase from Escherichia coli
Authors: Anderson Clare M. , Gray John
Published: July 2002

Leader peptidase from Escherichia coli was able to process the precursor of pea cytochrome ƒ synthesised in vitro. N‐Terminal sequencing established that cleavage by leader peptidase generated the same mature sequence as in pea chloroplasts. Processing by leader peptidase was much more efficient co‐translationally rather than post‐translationally, and the extent of post‐translational processing declined with time suggesting that the cytochrome ƒ precursor folded to an uncleavable conformation. Detergent extracts of pea thylakoid membranes were unable to process the cytochrome ƒ precursor co‐ or post‐translationally.
Functional size analysis of pyrophosphatase from Rhodospirillum rubrum determined by radiation inactivation
Authors: Wu Jia Jiu , Ma Jing Tyan , Pan Rong Long
Published: July 2002

A radiation inactivation technique was employed to determine the functional size of pyrophosphatase (PPase) from the chromatophores of Rhodospirillum rubrum. The activities of hydrolysis and synthesis reactions of pyrophosphatase and its coupled proton translocation decayed in a simple exponential function with the increase of radiation dosages. D
3, values of 5.2 ± 0.7 and 5.8 ± 0.8 Mrads were obtained for pyrophosphate hydrolysis and its associated proton translocation yielding molecular masses of 167.7 ± 30.7 and 156.3 ± 26.6 kDa, respectively. Similarly, a D
3, value of 4.4 ± 0.6 Mrads was measured for the acid‐base induced pyrophosphate synthesis resulting in a radiation sensitive size of 196.3 ± 31.9 kDa.
Several forms of chromaffin granule cytochrome b‐561 revealed by EPR spectroscopy
Authors: Burbaev D.Sh. , Moroz I.A. , Kamenskiy Yu.A. , Konstantinov A.A.
Published: July 2002

Low‐temperature EPR spectra of chromaffin granule membranes from bovine adrenal medulla reveal 3 different signals of the ferrie cytochrome b‐561. A typical
x
signal of a low‐spin cytochrome observed at g ∼ 3 is compromised of a high‐potential component with gz
= 3.14 and a low‐potential one with gx
= 3.11, the potential signal showing significantly faster relaxation. In addition, a highly temperature‐sensitive heme signal at g = 3.7 is observed which is fully retained in the preparation of granule membranes with b‐561 reduced by 50% but disappears upon full reduction of the cytochrome by ascorbate. The signal is strikingly similar to that of the mitochondrial low‐potential cytochrome b heme (b
L or b‐566). The presence of several forms of b‐561 in chromaffin granule membranes may provide a structural basis for the transmembrane electron transfer believed to be catalyzed by this hemoprotein.
Distribution of a negative regulator of haematopoietic stem cell proliferation (AcSDKP) and thymosin, β4 in mouse tissues
Authors: Pradelles Philippe , Frobert Yveline , Créminon Christophe , Ivonine Hélène , Frindel Emilia
Published: July 2002

A competitive enzyme immunoassay using acetylcholinesterase as tracer for thymosin β4, has been developed. Using this assay and a previously described EIA for AcSDKP, a negative regulator of pluripotent haematopoietic stem cell proliferation, the levels of these two peptides were determined in mouse tissue extracts. The combination of EIAs with different HPLC procedures validated these methods and clearly demonstrated the ubiquity of these peptides in mouse tissues. Similar results are reported for rabbit thymus which suggest different hypotheses for AcSDKP biosynthesis.
Two‐stage thermal unfolding of [Cys55]‐substituted Cro repressor of bacteriophage λ
Authors: Gitelson G.I. , Griko Yu.V. , Kurochkin A.V. , Rogov V.V. , Kutyshenko V.P. , Kirpichnikov M.P. , Privalov P.L.
Published: July 2002

It has been shown by scanning calorimetry and 1H NMR spectroscopy that thermal denaturation of mutant λ phage cro repressor in which Val55 was substituted for Cys, proceeds in 2 stages in contrast to the wild type protein. At neutral pH values, an additional cooperative transition has been observed at about 100°C. Calorimetric measurements on the mutant and its tryptic fragment lead to the conclusion that the two‐stage character of thermal unfolding of the mutant is due to a disruption of an additional cooperative domain in the dimer molecule which is stabilized by the S–S crosslink.
Computational approach towards the three‐dimensional structure of E. coli tyrosine aminotransferase
Authors: Jäger J. , Solmajer T. , Jansonius J.N.
Published: July 2002

We present a new model for E. coli tyrosine aminotransferase based on the X‐ray structures of the wild type and Val39Leu mutant of E. coli aspartate aminotransferase and computer simulation studies. Active site characteristics of the model are correlated with experimental observations on the specificity of these enzymes towards aromatic/dicarboxylic acid substrates.
Human urokinase contains Ga1NAcβ(1‐4)[Fucα(1‐3)]G1cNAcβ(1‐2) as a novel terminal element in N‐linked carbohydrate chains
Authors: Bergweff Aldert A. , Thomas-Oates Jane E. , van Oostrum Jan , Kamerling Johannis P. , Vliegenthart Johannes F.G.
Published: July 2002

Structural analysis of enzymically released N‐linked carbohydrate chains of human urokinase (urinary‐type plasminogen activator) by 1H NMR spectroscopy and FAB‐MS demonstrated that the N‐linked oligosaccharides on the only N‐glycosylation site contain diantennary structures with the novel Ga1NAcβ(1‐4)[Fucα(1‐3)]G1cNAcβ(1‐2) element in the upper or the lower branch.
Soluble interferon‐α receptor molecules are present in body fluids
Authors: Novick D. , Cohen B. , Rubinstein M.
Published: July 2002

Soluble forms of the interferon‐α receptor (sIFN‐αR) were identified in human serum and urine by Western blotting with monoclonal antibodies (MAb) directed against IFN‐αR, and by immunoprecipitation (Iptn) of a covalently cross‐linked complex of IFN‐αR and [125I]IFN‐α with anti IFN‐α MAb. Elevated levels or sIFN‐αR were found in sera of hairy cell leukemia patients. The soluble receptor from serum migrated as a 55 kDa protein in SDS‐PAGE, and, as expected, the cross‐linked product migrated as a 75 kDa protein. The soluble receptor from urine was found to be a protein of mol. wt. 45 kDa and its cross‐linked complex migrated as a 65 kDa protein. The calculated mol. wt. of the entire extracellular domain of the IFN‐αR prior to post‐translational modifications is 47,000. Since there are 12 potential glycosylation points in this extracellular domain, its actual mol. wt. may be its high as 70,000 Da. It is therefore concluded that sIFN‐αR molecules, corresponding to truncated forms of the extracellular domain of the cell surface IFN‐αR, are present in human serum and in normal human urine.
Differential sensitivity of membrane‐associated pyrophosphatases to inhibition by diphosphonates and fluoride delineates two classes of enzyme
Authors: Baykov Alexander A. , Dubnova Elena B. , Bakuleva Natalia P. , Evtushenko Olga A. , Zhen Rui-Guang , Rea Philip A.
Published: July 2002

1,1‐Diphosphonate analogs of pyrophosphate, containing an amino or a hydroxyl group on the bridge carbon atom, are potent inhibitors of the H+‐translocating pyrophosphatases of chromatophores prepared from the bacterium Rhodospirillum rubrum and vacuolar membrane vesicles prepared from the plant Vigna radiata. The inhibition constant for aminomethylenediphosphonate, which binds competitively with respect to substrate, is below 2 μM. Rat liver mitochondrial pyrophosphatase is two orders of magnitude less sensitive to this compound but extremely sensitive to imidodiphosphate. By contrast, fluoride is highly effective only against the mitochondrial pyrophosphatase. It is concluded that the mitochondrial pyrophosphatase and the H+‐pyrophosphatases of chromatophores and vacuolar membranes belong to two different classes of enzyme.
Primary structure of the 5 S subunit of transcarboxylase as deduced from the genomic DNA sequence
Authors: Thornton Charles G. , Kumar Ganesh K. , Shenoy Bhami C. , Haase F.Carl , Phillips Nelson F.B. , Park Vicki M. , Magner William J. , Hejlik Daniel P. , Wood Harland G. , Samols David
Published: July 2002

Transcarboxylase from Propionibacterium shermanii is a complex biotin‐containing enzyme composed of 30 polypeptides of three different types. It is composed of six dimeric outer subunits associated with a central cylindrical hexameric subunit through 12 biotinyl subunits; three outer subunits on each face of the central hexamer. Each outer dimer is termed a 5 S subunit which associates with two biotinyl subunits. The enzyme catalyzes a two‐step reaction in which methylmalonyl‐CoA and pyruvate form propionyl‐CoA and oxalacetate, the 5 S subunit specifically catalyzing one of these reactions. We report here the cloning, sequencing and expression of the monomer of the 5 S subunit. The gene was identified by matching amino acid sequences derived from isolated authentic 5 S peptides with the deduced sequence of an open reading frame present on a cloned P. shermanii genomic fragment known to contain the gene encoding the 1.3 S biotinyl subunit. The cloned 5 S gene encodes a protein of 519 amino acids, M
r, 57,793. The deduced sequence shows regions of extensive homology with that of pyruvate carboxylase and oxalacetate decarboxylase, two enzymes which catalyze the same or reverse reaction. A fragment was subcloned into pUC19 in an orientation such that the 5 S open reading frame could be expressed from the lac promoter of the vector. Crude extracts prepared from these cells contained an immunoreactive band on Western blots which co‐migrated with authentic 5 S and were fully active in catalyzing the 5 S partial reaction. We conclude that we have cloned, sequenced and expressed the monomer of the 5 S subunit and that the expressed product is catalytically active.
Characterisation of biotinylated liposomes for in vivo targeting applications
Authors: Loughrey Helen C. , Ferraretto Anita , Cannon Ann-Marie , Acerbis Giulia , Sudati Francesco , Bottiroli Giovanni , Masserini Massimo , Soria Marco R.
Published: July 2002

Liposomes containing monosialoganglioside (GM1) or polyethylene glycol (PEG) lipid derivatives have prolonged circulation in the blood. This favours liposome extravasation to tumour sites. In this report it is shown that inclusion of GM1, PEG550‐DPPE or PEG2000‐DPFE in liposomes containing biotin‐DPPE significantly diminished the ability of vesicles to bind to streptavidin in vitro. Steric inhibition due to the bulky head group of these lipids was least for biotin‐DPPE liposomes containing GM1. Biodistribution studies in C26 tumour‐bearing mice showed that GM1‐liposomes containing small amounts of biotin‐DPPE have long circulation life‐times in the blood. Using fluorescent microscopic techniques, liposomes containing both GM1 and biotin‐DPPE were detected within extra‐vascular spaces in tumours. In addition it was shown that biotin‐DPPE in GM1‐liposomes bound streptavidin in situ. These results suggest that GM1‐liposomes containing biotin‐DPPE have potential use as diagnostic or therapeutic reagents in pre‐targeting applications dependent on the high‐affinity interaction of biotin with streptavidin.
Stereo‐specific inhibition of sea urchin envelysin (hatching enzyme) by a synthetic autoinhibitor peptide with a cysteine‐switch consensus sequence
Authors: Nomura Kohji , Suzuki Norio
Published: July 2002

Inhibition of envelysin, a metalloproteinase which dissolves the fertilization envelope of sea urchin embryo, was studied using a synthetic autoinhibitor peptide, Ac‐Pro‐Arg‐Cys‐Gly‐Val‐Pro‐Asp‐Val‐NH2, with a ‘cysteine‐switch’ consensus sequence. Although its effect is reversible, the hatching of sea urchin embryos was effectively delayed by 0.5 mM of the peptide. When α1‐proteinase inhibitor was used as the substrate, envelysin was inhibited by the autoinhibitor and an Ala6 analogue, but not by a d‐Cys3 analogue. However, envelysin was weakly inhibited by both d‐ and l‐cysteines to the same extent. Snake venom α‐protease exhibited cleavage and inhibition behavior similar to envelysin with a little weaker stereo‐specificity. The results suggest that the coordination of the autoinhibitor Cys residue with the envelysin active site Zn is established only after the amino acid residues on both sides of the Cys residue get into an appropriate interaction with the catalytic site residues, and that the precise orientation of the cysteine SH group is essential. By contrast, thermolysin was weakly inhibited by the three peptide non‐stereo‐specifically. Furthermore, thermolysin cleaved the autoinhibitor at the Cys3 Gly4 bond when incubated without substrate.
Antisense oligonucleotides made of 2'‐O‐alkylRNA: their properties and applications in RNA biochemistry
Authors: Lamond Angus I. , Sproat Brian S.
Published: July 2002

Oligo(2'‐O‐alkylribonucleotides) have been developed recently as novel oligonucleotide analogues with properties that enhance their use as antisense probes. They possess high chemical stability and are resistant to hydrolysis by DNA‐ or RNA‐specific nucleases. Many forms of oligo(2'‐Oalkylribonucleotides) hybridise specifically and efficiently to complementary RNA sequences, forming stable duplexes that are not substrates for cleavage by RNase H. In combination with prosthetic reporter groups, such as biotin, DNP or fluorophores, oligo(2'‐O‐alkylribonucleotides) have important applications in a wide range of biochemical studies on RNA function and structure.
Minor‐groove binders are inhibitors of the catalytic activity of DNA gyrases
Authors: Störl K. , Störl J. , Zimmer Ch. , Lown J.W.
Published: July 2002

Non‐intercalating DNA minor‐groove binders may effectively inhibit the supercoiling activity of gyrases by influencing the enzyme recognition and cleavage site on DNA. For gyrase from Streptomyces noursei a wide range of inhibitory potency for different classes of ligands is observed. This can be explained by a number of structural and binding factors of the ligands competing with the gyrase on the target site of DNA, the mechanism of which is different from the classical gyrase inhibitors.
Autostimulation of the DnaK (HSP 70) ATPase of Escherichia coli
Authors: Richarme Gilbert , Kohiyama Masamichi
Published: July 2002

The ATPase activity of DnaK, the 70‐kDa chaperone of Escherichia coli, is stimulated by an unfolded protein. However, the stimulation of the DnaK ATPase by unfolded bovine pancreatic trypsin inhibitor can only be observed at low DnaK protein concentrations. At higher DnaK concentrations, the ATPase activity of DnaK cannot be stimulated by the addition of unfolded bovine pancreatic trypsin inhibitor. This is a consequence of the autostimulation of the DnaK ATPase at higher DnaK concentrations. The autostimulation of DnaK is reflected by a non‐linear dependence of ATP hydrolysis on DnaK concentration. Furthermore, DnaK exists as a mixture of monomers and dimers in equilibrium, and the dimers dissociate into monomers in the presence of ATP.
Increased expression of type VI collagen genes in drug‐induced gingival enlargement
Authors: Shikata Hideo , Utsumi Nobuo , Shimojima Takahiro , Oda Yoshio , Okada Yasunori
Published: July 2002

Fibrotic gingival enlargements induced by phenytoin or nifedipine were examined with special reference to type VI collagen expression. Immuno‐localization studies showed abnormal accumulation of type VI collagen around the collagen fiber bundles in the fibrotic gingival enlargements. Examination of total RNA extracted from fibroblasts and tissues of enlarged gingivae demonstrated increased type VI collagen steady‐state mRNA levels. These results suggest that excessive deposition of type VI collagen in drug‐induced gingival enlargement is attributed to increased expression of the collagen genes.
Genetic transfer of endothelin converting enzyme activity to CHO‐K1 cells: detection of positive cells by reverse hemolytic plaque assay
Authors: Shiraki Takuma , Sawamura Tatsuya , Ikura Tsuyoshi , Kobayashi Shigeo , Miwa Soichi , Masaki Tomoh
Published: July 2002

We have established a novel method of molecular cloning of endothelin converting enzyme, a key enzyme in the production of a potent vasoconstrictor endothelin‐1, by modification of the reverse hemolytic plaque assay. Also, we demonstrated that a cell line, CHO‐K1, showed no detectable activity of endothelin converting enzyme. This cell line was transfected with a cDNA library of bovine endothelial cells. The modified reverse hemolytic plaque assay was shown to detect even a single CHO‐K 1 cell that was changed to produce mature ET‐1 by transfection. Thus, this novel method is suggested to be useful for the molecular cloning of other secreted antigens and their processing enzyme.
A genetic approach to elucidating eukaryotic iron metabolism
Authors: Klausner Richard D. , Dancis Andrew
Published: July 2002

Studies of mutants of the yeast Saccharomyces cerevisiae have to led to the identification of genes required for high affinity iron uptake. Reduction of iron (III) outside the cell is accomplished by means of reductases encoded by FRE1 and FRE2, homologues of the gp91‐phox component of the oxygen reductase of human granulocytes. High affinity iron (II) transport from the exterior to the interior of the cell occurs by means of a transport system that has not been molecularly characterized. However, the transport process requires the activity of a copper‐containing oxidase encoded by FET3. The amino acid sequence of this protein resembles other multi‐copper oxidases, including mammalian ceruloplasmin. High affinity copper uptake mediated by the copper transport protein encoded by CTR1 is required to provide the FET3 protein with copper, and thus copper uptake is indirectly required for ferrous iron uptake. These genetic elements of yeast and their relationships may be conserved in complex eukaryotic organisms.
Expression patterns of DNA replication enzymes and the regulatory factor DREF during Drosophila development analyzed with specific antibodies
Authors: Yamaguchi Masamitsu , Hirose Fumiko , Nishimoto Yoshio , Naruge Toyobumi , Ikeda Masako , Hachiya Takahisa , Tamai Katsuyuki , Kuroda Kazufumi , Matsukage Akio
Published: July 2002

Summary— Specific antibodies were prepared against Drosophila DNA polymerase e and DREF, a regulatory factor for DNA replication‐related genes. Using these antibodies together with those for DNA polymerase α and proliferating cell nuclear antigen (PCNA), we examined expression patterns and sub‐cellular distributions of these proteins during Drosophila development. DNA polymerase α, ε and PCNA proteins were maternally stored in unfertilized eggs and maintained at high levels during embryogenesis. With distinct nuclear localization, proteins were observed in embryos at interphase stages throughout the 13 nuclear division cycles, suggesting that they all participate in rapid nuclear DNA replication during these cycles. In contrast, maternal storage of a DREF protein was relatively low and its level increased throughout embryogenesis. Strong nuclear staining with the anti‐DREF antibody was not observed until the nuclear division cycle 8. Immunostaining of various larval tissues from transgenic flies carrying the PCNA gene promoter‐lacZ fusion gene revealed co‐expression of DREF, PCNA and lacZ, suggesting that DREF regulates the expression of PCNA gene in these tissues. In addition, we detected a relatively high level of DREF in adult males as well as females. Since DNA polymerase α, ε and PCNA are hardly detectable in adult males, DREF very likely regulates genes other than those closely linked to DNA replication in adult males.
Rad24 is essential for proliferation of diploid cells in fission yeast
Authors: Tanaka Yoshimi , Okuzaki Daisuke , Yabuta Norikazu , Yoneki Takahiro , Nojima Hiroshi
Published: July 2002

The rad24
+ gene of Schizosaccharomyces pombe encodes a ubiquitously expressed 14‐3‐3 protein. We report here that Δrad24 cells displayed a defect in diploid colony formation, although they conjugated efficiently. We found that a cumulative deletion of mei2
+ gene almost completely suppressed this defect, and demonstrated using two‐hybrid analysis that Rad24 protein directly associates with Mei2 protein by recognizing Ser‐438 which is a phosphorylation target of Pat1 kinase. We conclude that constitutive progression to meiosis, caused by lack of Mei2 inhibition due to the absence of Rad24 protein, is the primary cause of the proliferative deficiency observed in Δrad24 cells.
Up‐regulation of gut‐enriched krüppel‐like factor by interferon‐γ in human colon carcinoma cells
Authors: Chen Zhi Y , Shie Jue-Lon , Tseng Chi-Chuan
Published: July 2002

Interferon‐γ (IFN‐γ) induces growth arrest and apoptosis of tumor cells but the mechanisms for these functions are unknown. Recently, gut‐enriched krüppel‐like factor (GKLF) was found to possess similar biological properties. Treatment of HT‐29 cells with IFN‐γ inhibited cell proliferation and induced apoptosis, the effect was found to associate with GKLF expression. IFN‐γ stimulates GKLF mRNA and protein levels in a dose‐ and time‐dependent manner and this process is independent of p53, occurs rapidly and does not require de novo protein synthesis indicating that GKLF is an immediate‐early IFN‐γ‐responsive gene. Moreover, overexpression of GKLF results in similar effect as IFN‐γ suggesting that GKLF may function as a downstream target of IFN‐γ.
Differential inheritance modes of DNA methylation between euchromatic and heterochromatic DNA sequences in ageing fetal bovine fibroblasts
Authors: Kang Yong-Kook , Koo Deog-Bon , Park Jung Sun , Choi Young-Hee , Lee Kyung-Kwang , Han Yong-Mahn
Published: July 2002

To elucidate overall changes in DNA methylation occurring by inappropriate epigenetic control during ageing, we compared fetal bovine fibroblasts and their aged neomycin‐resistant versions using bisulfite–PCR technology. Reduction in DNA methylation was observed in euchromatic repeats (18S‐rRNA/art2) and promoter regions of single‐copy genes (the cytokeratin/β‐lactoglobulin/interleukin‐13 genes). Contrastingly, a stable maintenance of DNA methylation was revealed in various heterochromatic sequences (satellite I/II/alphoid and Bov‐B). The differential inheritance mode of DNA methylation was confirmed through the analysis of individual neomycin‐resistant clones. These global, multi‐locus analyses provide evidence on the tendency of differential epigenetic modification between genomic DNA regions during ageing.
Distribution of blue‐sensitive photoreceptors in amphibian retinas
Authors: Takahashi Yusuke , Hisatomi Osamu , Sakakibara Shunsuke , Tokunaga Fumio , Tsukahara Yasuo
Published: July 2002

Previously, we reported that an opsin (Rc‐MS) belonging to the SWS2 group opsins is expressed in bullfrog green rods [Hisatomi, O. et al., FEBS Lett., 1999, 447, 44–48]. An anti‐Rc‐MS antiserum recognized the cones of the Japanese common newt, Cynops pyrrhogaster, which has no green rods. We isolated a cDNA encoding an SWS2 group opsin (Cp‐SWS2) from this newt and found that Cp‐SWS2 is expressed in a small population of the cones. Our results suggest that SWS2 opsins can be expressed in either green rods or cones of caudata. It seems reasonable to suppose that green rods arose before amphibia were divided into caudata and anura.
Membrane topology distinguishes a subfamily of the ATP‐binding cassette (ABC) transporters
Authors: Tusnády Gábor E , Bakos Éva , Váradi András , Sarkadi Balázs
Published: July 2002

A group of ATP‐binding cassette (ABC) transporters, including the yeast cadmium transporter (YCF1), the mammalian multidrug resistance‐associated protein (MRP), the multispecific organic anion transporter and its congener (MOAT and EBCR), as well as the sulfonylurea receptor (SUR), group into a subfamily by sequence comparison. We suggest that these MRP‐related proteins are also characterized by a special, common membrane topology pattern. The most studied ABC transporters, the cystic fibrosis transmembrane conductance regulator (CFTR) and the multidrug resistance (MDR) proteins, were shown to contain a tandem repeat of six transmembrane helices, each set followed by an ATP‐binding domain. According to the present study, in contrast to various membrane topology predictions proposed for the different MRP‐related proteins, they all seem to have a CFTR/MDR‐like core structure, and an additional, large, N‐terminal hydrophobic region. This latter domain is predicted to contain 4–6 (most probably 5) transmembrane helices, and is occasionally glycosylated on the cell surface. Since all the MRP‐related transporters were shown to interact with anionic compounds, the N‐terminal membrane‐bound domain may have a key role in these interactions.
Analysis of the contribution of the hinge region of human neutrophil collagenase (HNC, MMP‐8) to stability and collagenolytic activity by alanine scanning mutagenesis
Authors: Knäuper Vera , Docherty Andrew J.P , Smith Bryan , Tschesche Harald , Murphy Gillian
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Modulation of the ER Ca2+ channel BCC1 from tendrils of Bryonia dioica by divalent cations, protons and H2O2
Authors: Klüsener Birgit , Boheim Günther , Weiler Elmar W
Published: July 2002

Electrical properties of the ER Ca2+ channel BCC1 from tendrils of Bryonia dioica were analyzed after incorporation of BCC1 into black lipid bilayers. Single channel current fluctuations were modulated by divalent cations, protons and H2O2. Whereas the channel is permeable for Ca2+, Ba2+ and Sr2+, its conductance is strongly reduced in solutions containing MgCl2. Cu2+ and Zn2+ are potent inhibitors of BCC1 in micromolar concentrations. The open channel conductance of BCC1 increases with acidification of the electrolyte solution. H2O2 shows strong inhibitory effects on BCC1. The channel is almost completely closed at submillimolar concentrations of H2O2. The effects of pH and H2O2 on channel properties are directional and affect BCC1 at the Ca exit side, but not on the entry site. Thus, cytosolic pH and H2O2 levels may play an important role in the modulation of the cytoplasmic free calcium concentration through BCC1.
Serine‐threonine protein kinase activity of Elm1p, a regulator of morphologic differentiation in Saccharomyces cerevisiae
Authors: Koehler Carla M , Myers Alan M
Published: July 2002

The Saccharomyces cerevisiae gene ELM1 regulates morphologic differentiation and its nucleotide sequence predicts a novel protein kinase. Elm1p was expressed in yeast and insect cells and purified. Elm1p displayed protein kinase activity in autophosphorylation assays and towards exogenous substrates. Serine and threonine residues were identified as the acceptors in these reactions. These data together with previous genetic analysis of ELM1 function indicate that phosphorylation on serine and/or threonine residues of a particular substrate or set of substrates by Elm1p is required for repression of the filamentous growth differentiation state.
Dramatic enhancement of the specific expression of the heart‐type fatty acid binding protein in rat brown adipose tissue by cold exposure
Authors: Daikoku Takiko , Shinohara Yasuo , Shima Atsushi , Yamazaki Naoshi , Terada Hiroshi
Published: July 2002

To understand the difference in energy metabolisms in brown (BAT) and white (WAT) adipose tissues, we examined the steady‐state transcript levels of the heart‐type and adipose‐type fatty acid binding proteins (H‐FABP and A‐FABP, respectively) by Northern blot analysis. The transcript of H‐FABP in rat BAT was increased about 100‐fold by cold exposure, whereas that in WAT was negligible, and was increased only slightly by cold exposure. The transcript of A‐FABP was observed in both BAT and WAT, the level being slightly greater in WAT. However, its transcript level was not affected by cold exposure in either adipose tissue. In addition, on treatment with norepinephrine (NE), transcript level of H‐FABP was elevated markedly but that of A‐FABP was not changed in rat brown adipocytes. Therefore, the stimulatory effect of cold exposure on the transcript of H‐FABP in BAT was concluded to be mediated by NE, like that of the uncoupling protein (UCP). Thus, the expressions of H‐FABP and UCP may be controlled by the same mechanism.
Structure and dynamic studies by NMR of the potent sweet protein monellin and a non‐sweet analog
Authors: Mizukoshi Toshimi , Kohmura Masanori , Suzuki Ei-ichiro , Ariyoshi Yasuo
Published: July 2002

Monellin, an intensely sweet protein and a non‐sweet analog in which the AspB7 in monellin has been replaced with AbuB7 were studied by NMR. The results of our investigations show that the 3‐dimensional structure of these two proteins are very similar indicating that the lack of the β‐carboxyl group in the AbuB7 analog is responsible for the loss of sweet potency. Selectively labeled monellin was prepared by solid‐phase peptide synthesis by incorporating 15N‐labeled amino acids into 10 key positions including AspB7. The internal mobility of these 10 key residues in monellin was estimated by the method of model‐free analyses and our NMR studies show that AspB7 is the most flexible of these 10 residues. The flexibility of the AspB7 side chain may be important for receptor binding.
Kinetics of the inhibition of mitochondrial respiration by NO
Authors: Koivisto Ari , Matthias Anita , Bronnikov Gennady , Nedergaard Jan
Published: July 2002

The kinetics of the inhibition of mitochondrial respiration by NO was examined in isolated mitochondria (here obtained from rat brown adipose tissue). The K
i of NO for the inhibition was ∼27 nM; the IC50 of NO increased in proportion to the square of an increase in O2 tension. The K
m of O2 for respiration was ∼16 μM; in the presence of NO, the dependence of respiration on O2 tension had a Hill coefficient of ∼2. The unusual kinetics is probably related to the ability of cytochrome c oxidase to use 2 NO or 1 O2 as electron acceptor. The interaction between NO and O2 in the control of respiration could be described by the formula V
O2(O2, NO)=V
O2max·([O2]2/((16 μM·(1+[NO]/27 nM))2+[O2]2)). Thus, the kinetics is such that respiration in the presence of physiological levels of NO is very sensitive to decreasing O2 tension.
The olfactory adenylyl cyclase type 3 is expressed in male germ cells
Authors: Defer Nicole , Marinx Olivier , Poyard Madeleine , Lienard Marie Odile , Jégou Bernard , Hanoune Jacques
Published: July 2002

Elements of the olfactory pathway, such as receptors, receptor‐desensitization machinery, and cyclic nucleotide‐gated channels, are expressed in male germ cells. Here we report the expression, in rat testis, of both adenylyl cyclase type 3 (AC3) and the olfactory G protein subunit, Gαolf. Both are expressed in the same sub‐population of germ cells, pachytene spermatocytes to spermatids, and in residual bodies. Neither AC3 nor Gαolfwas found in Sertoli or in peritubular cells, as shown by Western blotting and immunocytochemical analyses. It thus appears that male germ cells contain all the elements of the signaling cascade present in olfactory cells.
NSF is required for the brefeldin A‐promoted disassembly of the Golgi apparatus
Authors: Fukunaga Takuya , Furuno Akiko , Hatsuzawa Kiyotaka , Tani Katsuko , Yamamoto Akitsugu , Tagaya Mitsuo
Published: July 2002

N‐Ethylmaleimide‐sensitive factor (NSF) is required for multiple pathways of vesicle‐mediated protein transport. Microinjection of a monoclonal anti‐NSF antibody almost completely blocked brefeldin A‐promoted Golgi disassembly without affecting the rapid release of β‐COP, a subunit of the Golgi coat proteins (COPI), from the Golgi apparatus. Similar results were obtained using a dominant‐negative NSF which is known to compete with endogenous NSF. The present results suggest that an NSF‐mediated step is present in the brefeldin A‐promoted disassembly of the Golgi apparatus.