bluuebunny/crossref_metadata_2025 huggingface dataset:
3′‐O‐Methylated analogs of 2–5A as inhibitors of virus replication
Authors: Sharma Opendra K. , Engels Joachim , Jager Alfred , Crea Roberto , van Boom Jacques , Goswami Biswendu B.
Published: July 2002

We have explored analogs of 2′–5′‐linked andeylic acid trimer (2–5A): 3′‐O‐methylated 2–5A, 2′‐end modified adenylate trimer with deoxyadenosine or araadenine, methyl phosphonate and methyl phosphorotriester analogs as potential antiviral agents. For the treatment of virus infections, 2–5A and its analogs may serve in lieu of interferon, however, the use of 2–5A has two serious limitations: it is presumed to be impermeable to most cells, and moreover, cellular enzymes rapidly degrade it. Methylated analogs of 2–5A core strongly inhibited virus growth when added directly to cells in culture. 2′‐End modified adenylate trimer with araadenine also inhibited virus growth, however, neither 2–5A nor other analogs showed any significant antiviral activity. The inhibition of virus growth was not due to the toxic effect of these compounds on cell growth as they had no inhibitory effect on the growth of uninfected cells
Selective inhibition of 5‐lipoxygenase by natural compounds isolated from Chinese plants, Artemisia rubripes Nakai
Authors: Koshihara Yasuko , Neichi Tomohiro , Murota Sei-itsu , Lao Ai-na , Fujimoto Yasuo , Tatsuno Takashi
Published: July 2002

Three of four natural compounds, which are caffeic acid, eupatilin and 4′‐demethyleupatilin, isolated from Chinese plant, Artemisia rubripes Nakai selectively inhibited 5‐lipoxygenase of cultured mastocytoma cells. Half‐inhibition doses (ID
50) for caffeic acid, eupatilin and 4′‐demethyleupatilin were 3.7, 14 and 18 × 10−6 M, respectively. The inhibition by caffeic acid was non‐competitive types. Prostaglandin synthase activities were little inhibited by eupatilin and 4′‐demethyleupatilin, but rather stimulated by affeic acid. The formation of leukotriene C4 and D4 by mast tumor cells was almost completely suppressed by these compounds at 10−4 M.
The effects of mutations, deletions and insertions of single amino acids on the three‐dimensional structure of globins
Authors: Kolchanov N.A. , Soloviov V.V. , Zharkikh A.A.
Published: July 2002

Using the method of calculation of low energy packings of α‐helices, we studied the effects of mutations, deletions and insertions of amino acids on the structure of sperm whale myoglobin. It was found that these events can cause complete breakdown of this structure.
Multiple environments of fluorinated anesthetics in intact tissues observed with 19F NMR spectroscopy
Authors: Wyrwicz Alice M. , Li Yao-En , Schofield John C. , Tyler Burt C.
Published: July 2002

The incorporation of two fluorine‐containing general anesthetic agents, halothane and methoxyflurane, into erythrocytes (from three different species), rabbit muscle and rabbit nerve, was followed with 19F NMR spectroscopy. Two major findings emerged from these studies: (1) multiple environments indicative of domain structure in the membrane can be observed depending on the anesthetic and the tissue type; and (2) the 19F chemical shifts of a given anesthetic were characteristic for the tissue examined. Halothane showed a single resonance in erythrocytes and multiple resonances in muscle and nerve, while methoxyflurane showed multiple resonances in both muscle and erythrocytes. The range of the 19F chemical shifts for the multiple peaks was as great as 6 ppm.
5‐Azacytidine increases the synthesis of embryonic hemoglobin (E2) in murine erythroleukemic cells
Authors: Zucker R.M. , Decal D.L. , Whittington K.B.
Published: July 2002

The addition of 5‐azacytidine to erythroleukemic cells which were induced to differentiate with DMSO or BA altered the expression of the hemoglobins. After the addition of 5‐azacytidine there was an increase in hemoglobin synthesis especially in the embryonic E2 band. The β‐globin increased in synthesis after 5‐azacytidine treatment. The level of hemoglobin synthesis in DMSO‐induced cells is less than BA‐induced cells while the effect of the 5‐azacytidine stimulation was greater with DMSO induction than with BA induction.
Isolation and amino acid sequence of a mating pheromone produced by mating type α cells of Saccharomyces kluyveri
Authors: Sakurai Akira , Sakamoto Mitsuhiro , Tanaka Hiroko , Esumi Yasuaki , Takahashi Nobutaka , Fujimura Hiroaki , Yanagishima Naohiko , Banno Isao
Published: July 2002

A peptide, termed αsk2 pheromone, was isolated from culture filtrates of mating type α cells of Saccharomyces kluyveri as a mating pheromone, having both shmoo inducing (in a cells of S. kluyveri and S. cerevisiae) and agglutinability inducing (in a cells of S. cerevisiae) actions. The amino acid sequence of αsk2 pheromone was determined as H—Trp—His—Trp—Leu—Ser—Phe—Ser—Lys—Gly—Glu—Pro—Met—Tyr‐OH by mass spectrometry, sequence analysis and enzymatic digestions.
Characterization of human haptoglobin cDNAs coding for α2FSβ and α1Sβ variants
Authors: van der Straten Ariane , Herzog Albert , Cabezón Teresa , Bollen Alex
Published: July 2002

A human liver library, derived from a heterozygous (Hp2‐1) donor, has been used to isolate cDNA clones coding for the haptoglobin (Hp) α1Sβ and α2FSβ variants. DNA sequencing has shown that the two variants are identical except for the αF duplicated segment in Hp α2FSβ. Four nucleotide changes have been found between the phenotypically different F and S regions of the Hp α2 gene, resulting in an Asp,Lys/Asn,Glu substitution.
Juvenile‐hormone‐binding protein from silk gland of Galleria mellonella
Authors: Wiśniewski J.R. , Kochman M.
Published: July 2002

Juvenile‐hormone‐binding protein (JHBP) has been detected in cytosol of Galleria mellonella silk glands. JHBP has been separated from juvenile hormone (JH) degrading enzymes using column chromatography on Sephacryl S‐300. JHBP has a sedimentation coefficient of 4.1 S, an isoelectric point of pH 4.7 and exhibits one class of binding sites (K
d JH III = 1.5 × 10−8 M) with the highest specificity towards JH II. It was found that Zwittergent 3–10 increases 10‐fold the JHBP activity and converts some higher molecular mass species of JHBP into a single, approx. 50‐kDa component.
The expression of genes encoding proteins of B800–850 antenna pigment complex and ribosomal RNA of Rhodopseudomonas capsulata
Authors: Klug Gabriele , Kaufmann Norbert , Drews Gerhart
Published: July 2002

The synthesis of the photosynthetic apparatus was induced in chemotrophically grown cultures of the wild type strain 37b4 of Rhodopseudomonas capsulata by lowering of oxygen partial pressure. In these induced but growth‐limited cultures the amount of total RNA per cell increased. Using specific DNA‐probes for genes of the light‐harvesting complex B800–850 and for ribosomal RNA it was shown that the levels of mRNA for the proteins of the light‐harvesting complex B800‐850 and for ribosomal RNA increased. In the mutant strain G1pho+, missing the B800–850 complex, but synthesizing small amounts of the M
r 10000 polypeptide of the complex, the B800–850‐specific mRNA was not increased during induction. It is concluded that the synthesis of the B800–850 complex is under transcriptional control and that, under these conditions, the level of rRNA also increased.
Solubilisation of cholesterol in human bile
Authors: Pattinson N.R.
Published: July 2002

Two non‐disruptive separation techniques (gel filtration, and CsCI density gradient ultracentrifugation) were used to characterise the mode of cholesterol transport in human bile. Both methods showed that biliary cholesterol is solubilised as a high −M
r, non‐micelle (lipoprotein) complex as well as the mixed micelle. The lipoprotein complex was separated between the densities 1.01 and 1.08, had a cholesterol to phospholipid ratio of 0.95 ± 0.41 and contained protein (7–20% by wt); The mixed micelle on the other hand sedimented at d ⩾ 1.18, and had a cholesterol to phospholipid ratio of 0.41 ± 0.13. Eighteen bile samples, obtained from either T‐tube, gallbladder or endoscopy, were analysed, and without exception they all contained a fraction of biliary cholesterol in the non‐micelle form. The results indicate that cholesterol is secreted, at least in part, as a lipoprotein complex independently of bile acids (mixed micelles).
γ‐Globin gene triplication and quadruplication in Japanese newborns
Authors: Harano K. , Harano T. , Kutlar F. , Huismar T.H.J.
Published: July 2002

Cord blood samples of 113 Japanese babies from Kurashiki, Japan, were analyzed for the presence of possible modifications in γ‐globin gene arrangements and for changes in the relative quantities of the γ chains Gγ, Aγ, and its variant AγT. As many as 9 babies had the γ‐globin gene triplication, one a γ‐globin gene quadruplication, and two a γ‐globin gene deletion. The triplication and quadruplication involved cross‐overs within the Gγ‐ and Aγ‐globin genes resulting in a hybrid gene with the 5'‐segment being derived from the Aγ and the 3'‐segment from the Gγ‐globin gene (‐AGγ‐). The Gγ value of the 9 babies with the ‐Gγ‐AGγ‐Aγ‐globin gene arrangement averaged 8̃0% and that of the baby with ‐Gγ‐AGγ‐AGγ‐Aγ‐ was 78%. Four of the 9 babies with the triplication and the baby with the quadruplication had an AγT mutation in trans; in these babies, the Aγ chain level (from the Aγ‐globin gene in 3'‐position in the tri‐ and quadruplication arrangements) was decreased to about one‐third and one‐sixth of the level observed in the simple AγT heterozygote. This observation supports the suggestion that transcription beginning at the 5'‐γ‐globin gene interferes with that of the 3'‐γ‐globin gene.
Selective photochemical reduction of either of the two bacteriopheophytins in reaction centers of Rps. sphaeroides R‐26
Authors: Robert Bruno , Lutz Marc , Tiede David M.
Published: July 2002

The bacterial photosynthetic reaction center (RC) contains two bacteriopheophytin (BPh) molecules, only one of which (characterized by Qx,y maxima at 540 and 760 nm) has been found to function in the normal photochemistry. We present here the formation of a new RC redox state, in which the apparently inactive BPh (Qx,y maxima at 530 and 752 nm) is selectively trapped in a reduced state by secondary photochemistry. This BPh is found to be associated to a bacteriochlorophyll (BChI), forming a BChl‐BPh complex analogous to the photochemically active BChl‐BPh acceptor complex. Electron transfer between the two BPhs is found not to occur.
A surface‐enhanced raman signal associated with functional manganese in oxygen‐evolving photosystem II membranes
Authors: Seibert Michael , Cotton Therese M.
Published: July 2002

O2 evolution
Manganese
Photosystem II
33‐kDa protein
34‐kDa protein
Photosynthesis
Purification of a 175‐kDa membrane protein, its localization in smooth and cardiac muscles
Authors: Koteliansky V.E. , Gneushev G.N. , Belkin A.M.
Published: July 2002

175‐kDa membrane protein
Vinculin
Smooth muscle
Cardiac muscle
Cytoskeleton‐membrane interaction
On the phase diagram of an L‐dipalmitoylphosphatidylcholine/cholesterol mixture
Authors: Koynova R.D. , Boyanov A.I. , Tenchov B.G.
Published: July 2002

L‐Dipalmitoylphosphatidylcholine
Cholesterol
Lipid
interaction
Phase
diagram
DSC
Two phases of inositol polyphosphate and diacylglycerol production at fertilisation
Authors: Ciapa Brigitte , Whitaker Michael
Published: July 2002

[3H]Inositol and [3H]arachidonic acid were used to label polyphosphoinositide phospholipids in sea urchin eggs. Both [3H]inositol polyphosphate (InsP3) and [3H]diacylglycerol (DAG) increase at fertilisation. An early increase in InsP3 occurs as the sperm‐induced calcium transient crosses the egg and exocytosis occurs; a later increase in InsP3, as calcium declines and the protein kinase C‐dependent Na/H antiporter causes the cytoplasmic pH to increase. These results support suggestions that a calcium‐induced hydrolysis of phosphatidylinositol bisphosphate occurs at fertilisation, that the production of diacylglycerol may be essential for exocytosis and that diacylglycerol production at fertilisation stimulates the Na/H antiporter. The increase in [3H]inositol polyphosphate as calcium declines indicates that this second messenger may have some function later in the cell cycle.
Functional domain of caldesmon
Authors: Szpacenko Adam , Dąbrowska Renata
Published: July 2002

Limted proteolysis of caldesmon has been used in studying the structure‐function relationship of this protein. Digestion with α‐chymotrypsin yields three major fragments of 110, 80 and 40 kDa. Only the 40 kDa fragment preserves functional properties of the parent molecule: it binds to F‐actin, causes inhibition of actomyosin ATPase and binds to calmodulin in a Ca2+‐dependent manner. Its further degradation produces an 18 kDa polypeptide that also retains all these properties. Neither F‐actin nor calmodulin binding induces dramatic changes in susceptibility to chymotryptic cleavage and the sites of cleavage of caldesmon.
Effects of thrombin, phorbol myristate acetate and prostaglandin D2 on 40–41 kDa protein that is ADP ribosylated by pertussis toxin in platelets
Authors: Halenda Stephen P. , Volpi Mario , Zavoico George B. , Sha'afi Ramadan I. , Feinstein Maurice B.
Published: July 2002

Intact platelets were stimulated with thrombin and the amount of GTP‐binding protein (G‐protein) oligomers was assessed by measuring ADP ribosylation of 40–41 kDa protein by pertussis toxin in isolated membranes. The toxin substrate fell by 57–62% in 10–60 s, but then returned towards normal over 5 min. Recovery was greatly enhanced by removal of thrombin from receptors with hirudin. Phorbol myristate acetate increased ADP‐ribosylatable protein, but only back to initial levels prior to PMA. In contrast prostaglandin D2 plus theophylline (which increase cyclic AMP) did not increase ADP ribosylation, but could completely block the fall of the toxin substrate caused by thrombin. These results indicate that activation of thrombin receptors promotes the dissociation of G‐protein oligomers to release free α‐subunits, and this effect can be modulated by protein kinase C and cyclic AMP‐dependent protein kinase. The possible relationships of these findings to the regulation of stimulus‐response coupling in platelets is discussed.
The effect of pH on the conformation and stability of the structure of plant toxin — ricin
Authors: Bushueva T.L. , Tonevitsky A.G.
Published: July 2002

The effect of pH on the conformation of ricin and its A‐ and B‐chains has been studied by measuring their intrinsic fluorescence. At pH 5.0 and 7.5, the structural stability of toxin and subunits was estimated according to the denaturing action of guanidine hydrochloride. It was demonstrated that the fluorescence of native toxin and catalytic A‐subunit does not depend significantly on pH in the range pH 3–8, whereas ricin B‐chain undergoes a structural transition at pH < 5.0. The structural stability of ricin and isolated chains differs significantly at pH 7.5 and 5.0; the structural stability of ricin and the A‐chain increases, whereas that of the B‐chain decreases.
A new 5′‐non‐coding region for human placental insulin‐like growth factor II mRNA expression
Authors: Le Bouc Y. , Noguiez P. , Sondermeijer P. , Dreyer D. , Girard F. , Binoux M.
Published: July 2002

A human placenta cDNA library was screened for insulin‐like growth factor II (IGF‐II). Four clones were selected, which exhibited an IGF‐II cDNA coding sequence identical to those previously described for human adult liver IGF‐II cDNA. Extensive sequence diversity was observed in the 5′‐non‐coding region, probably resulting from differential intron splicing.
A 32‐kDa protein associated with phospholipase A2‐inhibitory activity from human placenta
Authors: Hayashi Hideki , Owada M.Koji , Sonobe Seiji , Kakunaga Takeo , Kawakatsu Hisaaki , Yano Junichi
Published: July 2002

Two monomeric 32‐kDa proteins, termed 32K‐I (pI 5.8) and 32K‐II (pI 5.1), were isolated from human placenta, which was solubilized by a Ca2+‐chelator. Only 32K‐I was associated with PLA2‐inhibitory activity. CNBr peptide mapping indicated that 32K‐I was distinct from 32K‐II and two 36‐kDa proteins, called calpactin I and II or lipocortin II and I, which have been shown to possess PLA2‐inhibitory activity. 32K‐I bound to PS in a Ca2+‐dependent manner. 32K‐I was detected in many tissues except brain, cardiac and skeletal muscle.
Epidermal and transforming growth factors modulate secretion of a 69 kDa phosphoprotein in normal rat kidney fibroblasts
Authors: Laverdure G.R. , Banerjee D. , Chackalaparampil I. , Mukherjee B.B.
Published: July 2002

Our study shows that the secretion of a major non‐glycosylated, phosphoprotein of 69 kDa (pp69) is a specific marker for non‐transformed NRK‐49F cells. Treatment of NRK‐49F cells with EGF alone or with different combinations of EGF plus TGF‐β modulates the secretion of pp69, suggesting its relationship with cellular proliferation. Antibody raised against pp69 recognizes, in addition to pp69, another major phosphoprotein of 62 kDa (pp62) secreted by RR1022 and spontaneously transformed NRK‐49F cells. Immunoprecipitation of total cell lysates from both NRK‐49F and RR1022 cells with anti‐pp69 antibody detected only pp69. These observations suggest a precursor‐product relationship between pp69 secreted by non‐transformed NRK‐49F cells and pp62 secreted by transformed cells.
Export and secretion of overproduced OmpA‐β‐lactamase in Escherichia coli
Authors: Bolla Jean-Michel , Lazdunski Claude , Inouye Masayori , Pagès Jean-Marie
Published: July 2002

The export of β‐lactamase to the periplasm of Escherichia coli can be directed by the OmpA signal peptide in the secretion cloning vector pIN‐III. The overproduction of the hybrid precursor specifically induces a delay in the onset of processing of newly synthesized polypeptide chains. However, when the processing starts, no alteration in the rate of cleavage itself is observed. Our results suggest that the temporal mode of processing (which reflects translocation) does not depend on the nature of the signal peptide but rather depends on the nature of the polypeptide chain exported.
The rate of oxygen consumption and superoxide anion formation by stimulated human neutrophils The effect of particle concentration and size
Authors: Green Monika J. , Hill H.Allen O. , Tew David G.
Published: July 2002

The respiratory burst of neutrophils was measured as a function of the ratio of the opsonised beads to neutrophils. The rate of oxygen uptake was found to be linear up to a bead:neutrophil ratio of 25. The production of the superoxide anion, as measured by the rate of reduction of cytochrome c, was negligible until a certain ‘critical’ value of the bead:neutrophil ratio was reached. The rate of oxygen consumption and superoxide release above the critical value varies linearly with the bead:neutrophil ratio. Both the rate of oxygen consumption and of superoxide release vary with the square of the radius of the ingested particle. It is suggested that this depends on the surface area of neutrophil membrane, activated by contact with the antagonist.
Purification and properties of an N‐acetylgalactosamine specific lectin from the plant pathogenic fungus Rhizoctonia solani
Authors: Vranken An M. , Van Damme Els J.M. , Allen Anthony K. , Peumans Willy J.
Published: July 2002

A lectin was isolated from Rhizoctonia solani mycelium by affinity chromatography on gum arabic‐Sepharose. It is a dimeric protein composed of 2 identical subunits of 13 kDa with high contents of asparagine/ aspartic acid, valine, glycine, glutamine/glutamic acid and lysine. The R. solani agglutinin (RSA) exhibits specificity towards N‐acetylgalactosamine, and preferentially agglutinates human type A over type B and type O erythrocytes.
Cloning, nucleotide sequence and transfer of genes for the B800–850 light harvesting complex of Rhodobacter sphaeroides
Authors: Ashby Mark K. , Coomber Shirley A. , Hunter C.Neil
Published: July 2002

The genes that encode the α and β polypeptides of the major light‐harvesting complex of Rhodobacter sphaeroides, B800–850, have been cloned and sequenced through the use of oligonucleotides based upon the known polypeptide sequences. These genes, pucA and B, are transcribed in the order B, A, are of 150 and 164 nucleotides respectively, and are separated by a spacer region of 14 nucleotides. Transfer of these genes to mutant M21 lacking the B800–850 complex has been accomplished and absorbance spectra of recombinant strains M2181 and M2184 show that expression of pucA and B is comparable to levels found in the wild type.
Agonist‐induced α1‐adrenergic receptor changes
Authors: Fratelli Maddalena , De Blasi Antonio
Published: July 2002

Short‐term receptor regulation by agonists is a well‐known phenomenon for a number of receptors but little is known about the regulation of α1‐adrenergic receptors. In the present study we provide evidence of α1‐adrenergic receptor changes induced by agonists on DDT1 MF‐2 smooth muscle cells. The cells were preincubated with the agonist and receptor changes were investigated in the cells washed free of the agonist. On cells pretreated with norepinephrine the number of receptors recognized by [3H]prazosin at 4°C was reduced by 38%. The receptors were not degraded as the number of sites was the same in control and norepinephrine‐treated cells when binding was measured at 37°C. When binding was measured on fragmented membranes (at 4°C), the number of receptors was the same in control and norepinephrine‐treated cells, suggesting that the disruption of cellular integrity might expose receptors which are probably sequestered after agonist treatment. We conclude that agonists induced rapid sequestration of receptors on intact DDT cells.
Unprecedented lysyloxidase activity of Pichia pastoris benzylamine oxidase
Authors: Tur Sigmund S. , Lerch Konrad
Published: July 2002

Benzylamine oxidase (EC 1.4.3.6) from the yeast Pichia pastoris is a 106 kDa quinoprotein containing one copper atom per molecule. It has a broad substrate specificity ranging from butylamine to peptidyl lysine in collagen and elastin. The kinetic data obtained using lysine‐containing model peptides as substrates indicate an astonishing similarity to mammalian lysyloxidase. This similarity is further supported by the inhibition of both enzymes with β‐aminopropionitrile.
Sequence of the gene encoding phosphoglycerate mutase from Saccharomyces cerevisiae
Authors: White Malcolm F. , Fothergill-Gilmore Linda A.
Published: July 2002

The gene encoding yeast phosphoglycerate mutase was isolated, and its sequence was determined. The gene specifies a protein of 246 amino acids, and contains no introns. The sequence shows a strong codon bias. The upstream untranslated portion of the gene contains a CT‐rich block such as is found in many highly expressed yeast genes, but does not have the associated CAAG sequence.
Role of peptide substrate structure in the selective processing of peptide prohormones at basic amino acid pairs by endoproteases
Authors: Gluschankof Pablo , Gomez Sophie , Lepage Agnès , Créminon Christophe , Nyberg Fred , Terenius Lars , Cohen Paul
Published: July 2002

Three putative processing enzymes, each with defined action in a prohormone system, a ‘pro‐ocytocin‐neurophysin convertase’ from bovine neurohypophysis secretory granules, a ‘Leu‐enkephalin Arg6 generating enzyme’ from human CSF and the endoprotease from the ‘S‐28 convertase’ complex of rat brain cortex, were tested for their ability to hydrolyze peptides deriving from pro‐ocytocin, pro‐enkephalin B and pro‐somatostatin, respectively at pairs of basic amino acids. The observations suggest that structural parameters specified by the peptide region around the dibasic moieties govern recognition by the enzyme and define which peptide bond is hydrolyzed.
Cell surface fibronectin of mouse peritoneal macrophages
Authors: Beppu Masatoshi , Masa Hideo , Kikugawa Kiyomi
Published: July 2002

Fibronectin (FN) was detected on thioglycollate‐induced mouse peritoneal macrophages by binding the 125I‐labeled F(ab′)2 fragment of rabbit anti‐human plasma fibronectin. The cell surface fibronectin (sFN) was removed from the surface of the macrophage monolayer by limited trypsinization. After trypsinization, binding of 125I‐labeled plasma fibronectin (125I‐pFN) to the macrophage monolayer was increased, suggesting that the FN receptor covered with sFN was exposed by trypsinization without destroying the receptor activity. The amounts of saturation binding of 125I‐pFN to the macrophage monolayers before and after trypsinization were about 2.4 and 6.3 μg per 106 cells, respectively, indicating that the macrophage monolayer has the capacity of binding 6.3 μg FN per 106 cells, and the FN receptor equivalent to about 4 μg pFN per 106 cells is covered with sFN.
Deprivation of nerve growth factor rapidly increases purine efflux from cultured sympathetic neurons
Authors: Tolkovsky Aviva M. , Anne Buckmaster E.
Published: July 2002

The efflux of [3H]purines from cultured sympathetic neurons prelabelled with [3H]adenine is accelerated 2–3‐fold within hours of nerve growth factor (NGF) withdrawal and is reduced by readdition of NGF. Addition of 8‐(4‐chlorphenyl‐thio) cAMP, which delays neurite degeneration, reduced the enhanced efflux of purines, as did the addition of cycloheximide, MgCl2 and the protease inhibitor tosyl‐L‐lysine chloromethyl ketone. Colchicine accelerated purine efflux and neurite degeneration but 2‐deoxyglucose increased purine efflux without inducing degeneration, suggesting that ATP reduction itself is not the cause of neurite degeneration. The increase in purine efflux is thus an early biochemical event that has diagnostic value for the study of NGF action since deprivation is detected well before irreversible changes become established.
Blockade of ADP‐induced Ca2+‐signal and platelet aggregation by lipoxygenase inhibitors
Authors: L. Borin M , Pinelis V.G. , Ivanova M.A. , Kudinov Yu.V. , Azizova O.A. , Markov C.M. , Khodorov B.I.
Published: July 2002

Stimulation of platelets results in the liberation of arachidonic acid (AA) which is further metabolized via the cyclooxygenase or lipoxygenase (LPG) pathway. We have examined the effect of inhibition of LPG on (i) the ADP‐induced increase of cytoplasmic Ca2+ concentration and (ii) platelet aggregation. Lipoxygenase inhibitors, nordigidroguaiaretic acid (NDGA) and BW‐755C, both suppressed ADP‐induced Ca2+‐signals and aggregation in a dose‐dependent manner, with an IC50 value of 1–2 μM for NDGA. Qualitatively the same effect was obtained with 4‐bromophenylacyl bromide, the inhibitor of phospholipases A2 and C. By contrast, cyclooxygenase inhibitor indomethacin had only a negligible effect on Ca2+‐signals and suppressed only the second phase of ADP‐induced aggregation. It is concluded that the LPG pathway of AA metabolism in platelets might play a crucial role in ADP‐induced Ca2+‐signal generation and platelet aggregation.
Purification to homogeneity and characterization of major fatty acid ethyl ester synthase from human myocardium
Authors: Bora Puran S. , Spilburg Curtis A. , Lange Louis G.
Published: July 2002

Non‐oxidative metabolism of ethanol via fatty acid ethyl ester synthase is present in those extrahepatic organs most commonly damaged by alcohol abuse. DEAE‐cellulose chromatography of human myocardial cytosol at pH 8.0 separated synthase I, minor and major activities, eluting at conductivities of 5,7 and 11 mS, respectively. The major synthase was purified 8900‐fold to homogeneity by sequential gel permeation, hydrophobic interaction, and anti‐human albumin affinity‐chromatographies with an overall yield of 2596. SDS‐PAOE showed a single polypeptide with a molecular mass of 26 kDa and gel permeation chromatography under nondenaturing conditions indicated a molecular mass of 54 kDa for the active enzyme. The purified enzyme catalyzed ethyl ester synthesis at the highest rates with unsaturated octadecanoic fatty acid substrates (V
max = 100 and 65 nmol/mg/h for oleate and linoleate, respectively). K
m values for oleate, linoleate, arachidonate, palmitate and stéarate were 0.22 mM, 0.20 mM, 0.13 mM, 0.18 mM and 0.12 mM, respectively. Thus, human heart fatty acid ethyl ester synthase (major form) is a soluble dimeric enzyme comprised of two identical, or nearly identical, subunits (M
r = 26000).
Identification of a potent synthetic HIV1 immunogen compromising gag‐P24 tandem T‐ and B‐cell epitopes
Authors: Chong Pele , Sia Charles , Sydor Margret , Klein Michel
Published: July 2002

Recent studies indicate that the gag gene products may play a crucial role in the immune response against HIV infection since clinical progression to AIDS is associated with a reduction in the level of circulating antibodies to gag p24 and antibodies raised against p 17 peptide can inhibit HIV1 infection in vitro. Using conventional structure prediction algorithms for T‐cell and B‐cell epitopes, we have selected and chemically synthesized several gag peptides. In particular, an unconjugated HIV1‐p24 peptide containing both B‐ and T‐cell epitopes in tandem plus Freund's adjuvant induced a strong antibody response in both mice and rabbits against p24 and its precursor p55 as judged by immunoblotting. In addition, the peptide presented in the appropriate MHC context was shown to be highly stimulatory for p24 specific murine T‐cell clones.
Purification and characterization of a tartrate‐sensitive acid phosphatase of Trypanosoma brucei
Authors: Schell Dietmar , Stierhof York-Dieter , Overath Peter
Published: July 2002

In search for invariant surface proteins in Trypanosoma brucei bloodstream forms, acid phosphatase was investigated. Earlier work had shown that part of the cellular phosphatase activity is associated with the flagellar pocket of the parasite. It is demonstrated that T. brucei contains at least two membrane‐bound enzymes, one is sensitive to the inhibitor L‐(+)‐tartrate while the other is resistant. The tartrate‐sensitive phosphatase was purified to homogeneity by monoclonal antibody affinity chromatography and shown to be a glycoprotein of low abundance (13,000 molecules/ cell). It has an apparent molecular weight of 70,000 Da. The usefulness of acid phosphatase as a marker for characterizing the membrane lining the flagellar pocket is discussed.
Calmodulin is a potent target for new hypothalamic neuropeptides
Authors: Horv́ath L. , Barkhudaryan N. , Galoyan A.A. , Ovádi J.
Published: July 2002

Recently, five glycopeptides with coronaro‐constrictory properties were isolated from bovine hypothalamus [(1988) Neurochemistry (USSR) 7, 519‐524]. Calmodulin has been recognized in our laboratory as a target protein for the neuropeptides isolated from hypothalamus. The results of indirect enzyme‐linked immunosorbent assay have shown that the new hypothalamic neuropeptides antagonize with the monospecific anti‐calmodulin antibody for Calmodulin binding although they are not fragments of Calmodulin. The inhibitory potency of the peptides is dependent on their concentration and the length of the polypeptide chain. Four out of five peptides are effective in nM concentration range. Ca2+ stimulates the binding of peptides to calmodulin; however, immunocomplex can be formed in the absence of Ca2+ as well. The effects of trifluoperazine and peptides on the calmodulin/antibody interaction are not additive, suggesting the cooperativity between the binding sites on calmodulin. Under physiological conditions the presence of the peptides could produce distinct conformers of calmodulin which may exhibit altered potency for stimulation/inhibition of target enzymes.
Isolation of a protein with cytokinin‐receptor properties by means of anti‐idiotype antibodies
Authors: Kulaeva O.N. , Karavaiko N.N. , Moshkov I.E. , Selivankina S.Yu. , Novikova G.V.
Published: July 2002

A protein from cytosol of 10‐day‐old barley leaves with cytokinin‐receptor properties was isolated and 12 000‐fold purified by anti‐idiotype antibodies from anti‐BA serum. Cytokinin‐binding properties of this protein were demonstrated by its competition with Abba for immobilized BA in competitive ELISA. In the presence of BA the protein activates in vitro rRNA synthesis in the transcription elongation system containing chromatinbound RNA‐polymerase 1 from barley leaves. The protein with similar properties was isolated from barley leaf cytosol with BA‐Sepharose. The control proteins isolated with adenine‐ and ethanolamine‐Sepharose did not possess cytokinin‐binding properties and had no effect on RNA synthesis in vitro.
Formation of tritium‐labeled polysialylated gangliosides in the cytosol of rat cerebellar granule cells in culture following administration of [3H]GM1 ganglioside
Authors: Chigorno Vanna , Valsecchi Manuela , Sonnino Sandro , Tettamanti Guido
Published: July 2002

GM1 ganglioside tritium‐labeled at C‐3 of sphingosine has been administered to rat cerebellar granule cells. Tritiated polysialylated gangliosides were observed in the cytosol of the cells, where they resulted in a higher amount after a short period of chase. This, together with the data showing an increase of the tritiated polysialylated gangliosides in the total particulate fraction in parallel to the prolonging of the chase period, suggests that cytosolic gangliosides could be a way of transporting neosynthesized gangliosides from the Golgi apparatus to the plasma membranes.
Epidermal growth factor and platelet‐derived growth factor promote translocation of phospholipase C‐γ from cytosol to membrane
Authors: Kim Uh-Hyun , Kim Hee-Sook , Rhee Sue Goo
Published: July 2002

Treatment of HER 14 cells with epidermal growth factor (EGF) or platelet‐derived growth factor (PDGF) induced a translocation of phospholipase C‐γ (PLC‐γ) from cytosol to membrane. In such growth factor‐treated cells, cytosolic PLC‐γ was found to contain more phosphotyrosine than membrane‐associated enzyme. Because these growth factors have been shown to promote both the physical association of PLC‐γ with their receptors and the subsequent phosphorylation of the enzyme directly by the membrane‐bound receptor tyrosine kinases, the membrane assocation of PLC‐γ may simply be due to the formation of transient enzyme (receptor)‐substrate (PLC‐γ) complexes. If this is the case, membrane‐associated PLC‐γ would be expected to be released from membrane after undergoing tyrosine phosphorylation. However, tyrosine phosphorylation of membrane‐associated PLC‐γ by the EGF receptor in vitro did not result in the release of PLC‐γ from membrane. Thus, the association of PLC‐γ with membrane would appear to involve more than enzyme‐substrate complex.
Cellular incorporation and localization of fluorescent derivatives of gangliosides, cerebroside and sphingomyelin
Authors: Huang Richard T.C. , Dietsch Ellen
Published: July 2002

Fluorescent dansyl derivatives of 3 natural sphingolipids (gangliosides, cerebroside und sphingomyelin) were shown to be readily taken up by culture cells (HeLa‐, MDCK‐ and primary rat brain cells). A part of the incorporated fluorescent sphingolipids remained associated with the cells after incubation in a culture medium containing serum, showing a cellular integration of these lipids. Microscopical studies indicated a localization of incorporated lipids in distinct subcellular regions; whereas dansyl cerebroside densely stained structures suggestive of the cytoskeleton and the actin filament, dansyl sphingomyelin and dansyl gangliosides were primarily associated with the plasma membrane. The findings are consistent with the current views on the arrangement of sphingolipids in animal cells.
NMR investigation of the interaction of mithramycin A with d(ACCCGGGT)2
Authors: Keniry Max A. , Banville Debra L. , Levenson Corey , Shafer Richard H.
Published: July 2002

The binding of mithramcyin A to d(ACCCGGGT)2 has been investigated by one‐ and two‐dimensional 1H NMR spectroscopy. Titration of the drug into the octamer solution results in loss of the oligonucleotide C2 symmetry at stoichiometric ratios less than 4 drug molecules per duplex. However, at a ratio of 4:1 (drug/duplex), the C2 symmetry of the oligonucleotide is restored. From these data it is evident that more than one complex forms at ratios less than 4:1 while only one complex predominates at the ratio 4:1. This is the first report of a DNA octamer which binds 4 large drug molecules. These results are compared to those we have recently reported for mithramycin binding to d(ATGCAT)2, where only a single, bound complex is observed, with a stoichiometry of 2:1.
Fast cell membrane displacements in B lymphocytes Modulation by dihydrocytochalasin B and colchicine
Authors: Mittelman Leonid , Levin Shlomo , Korenstein Rafi
Published: July 2002

A novel type of cell membrane movement was characterized in B lymphocytes. Local submicron cell membrane displacements, within the frequency range 0.3–15 Hz, were registered in a murine lymphoma B cell line by a novel optical method based on point dark field microscopy. The cell membrane displacements were measured by monitoring changes in light scattering from very small illuminated areas (0.25 μm2) at the edge of the cell surface. B lymphocytes manifest a relative change in light scattering of 7.7 ± 1.3% (mean ± SD) which corresponds to cell membrane transverse displacement of 131 ± 22 nm. The confinement of cell membrane displacements to microdomains (≤0.2 μm2) emerged from the observed dependence of the displacement amplitude on the area size from which it is monitored. Colchicine (1 μM) decreased membrane fluctuations down to a value of 88 ± 14 nm, whereas dihydrocytochalasin B (2 μM) increased the amplitude of membrane displacements up to 184 ± 31 nm. These findings demonstrate the existence of a dynamic mechanical interaction between the cytoskeleton and the cell membrane in the frequency range of 0.3–15 Hz. The modulation of these interactions by the disruption of microfilaments or microtubules is explained in terms of the induced strain changes imposed on the cell membrane.
A dysfunctional Cl inhibitor protein with a new reactive center mutation (Arg‐444→Leu)
Authors: Frangi Donatella , Aulak Kulwant S. , Cicardi Marco , Harrison Richard A. , Davis Alvin E.
Published: July 2002

A Pl mutation (Arg‐444→Leu) was identified in a dysfunctional Cl inhibitor from a patient with type 2 hereditary angioneurotic edema. The mutation was defined at the level of the protein (by sequence analysis of the Pseudomonas aeruginosa elastase‐derived reactive center peptide), and the mRNA (CGC→CTC) (by sequence analysis of PCR‐amplified DNA).
Multiple mitochondrial DNA deletions in an elderly human individual
Authors: Zhang Chunfang , Baumer Alessandra , Maxwell Ronald J. , Linnane Anthony W. , Nagley Phillip
Published: July 2002

We have used the polymerase chain reaction (PCR) to study deletions in the mitochondrial DNA (mtDNA) of an elderly human individual. An extended set of PCR primers has been utilised to identify 10 mitochondrial DNA deletions in a 69‐year‐old female subject with no known mitochondrial disease. The particular deletions visualised as PCR products depended on the primer pairs used, such that the more distantly separated PCR primers enabled visualisation of larger deletions. Some deletions were common to the heart, brain and skeletal muscle, whereas others were apparently specific to individual tissues. DNA sequencing analysis of PCR products showed that short direct repeat sequences (5 to 13 bp) flanked all deletion breakpoints; in most cases one copy of the repeat was deleted. It is proposed that the accumulation of such multiple deletions is a general phenomenon during the ageing process.
Separation of fibronectin from a plasma gelatinase using immobilized metal affinity chromatography
Authors: Smilenov Lubomir , Forsberg Erik , Zeligman Ilia , Sparrman Marianne , Johansson Staffan
Published: July 2002

Conventional preparations of plasma fibronectin are known to contain a co‐purifying gelatinase [1986, J. Biol. Chem. 261, 4363–4366], but so far useful methods to remove the protease have not been available. In this study a number of different methods were tested in order to achieve separation of the two proteins. Immobilized metal affinity chromatography was found to be efficient for this purpose, and a convenient procedure to separate the two proteins under nondenaturing conditions on chelating Sepharose charged with Co2+, Ni2+, or Zn2+ is described. An alternative method employing pH gradient elution of an Fe3+ gel also resolved fibronectin from the gelatinase. The Fe3+ gel bound both proteins at pH 6.0 but not at pH 7.4, suggesting that the two proteins were phosphorylated. The described procedures will now allow studies of the functions of fibronectin in the absence of the contaminating protease.
The His‐probe method: Effects of histidine residues introduced into the complementarity‐determining regions of antibodies on antigen‐antibody interactions at different pH values
Authors: Ito Wataru , Sakato Nobuo , Fujio Hajime , Yutani Katsuhide , Arata Yoji , Kurosawa Yoshikazu
Published: July 2002

We examined the effects or histidine residues that were artificially introduced into complementarity‐determining regions of antibodies on antigen‐antibody Interactions at different pH values. Using a monoclonal antibody specific ror hen egg‐white lysozyme and three mutant antibodies that contained a histidine residue. we measured binding constants for antibodies and lysozyme at different pH values (PH 5–8). No gross conformational changes were evident over this range of pH vulues, as determined by analysis of the spectra of circular dichroism. Since the charge on a histidine residue is the most likely factor that can vary over this range of pH values, differences on pH‐dependent antigen‐binding patterns observed between the wild‐type and mutant antibodies should be due mainly to the effects of the charges on the histidine residues. The three mutant antibodies showed different and characteristic patterns of pH‐dependent binding to lysozyme, which depended on the location of the artificially introduced histidine residues.
Do unconventional myosins exert functions in dynamics of membrane compartments?
Authors: Coudrier Evelyne , Durrbach Antoine , Louvard Daniel
Published: July 2002

Unconventional myosins have now been identified in amoeba as well as in higher eucaryotic cells. Their cellular localization, their ability to bind membrane vesicles and their ability to produce in vitro movement suggest that they can generate forces on the plasma membrane relative to actin filaments as well as on membrane compartments relative to actin. Genetic approaches and biochemical analysis of cells over‐producing nonfunctional domains of unconventional myosins have provided direct evidence for a role of unconventional myosins in movement of intracellular vesicles and have allowed us to formulate hypotheses about the possible mechanisms by which unconventional myosins could participate in the intracellular transport of membrane proteins and secretary proteins.
Conformation of two non‐immunosuppressive FK506 analogs when bound to FKBP by isotope‐filtered NMR
Authors: Petros Andrew M. , Kawai Megumi , Luly Jay R. , Fesik Stephen W.
Published: July 2002

The 3D structure of two unlabeled FK506 analogs, (R)‐ and (S)‐[18‐OH]ascomycin, when bound to [U‐13C,14N]FKBP were determined by isotope‐filtered 2D NMR experiments. The structures for the R and S isomers that bind tightly to FKBP but lack immunosuppresive activity are compared to each other and to the conformation of the potent immunosuppressant, ascomycin, when bound to FKBP. The results are interpreted in terms of calcineurin binding to the FKBP/ascomycin complex.
The influence of oligodeoxyribonucleotide phosphorothioate pyrimidine strands on triplex formation
Authors: Kim Sung-Gug , Tsukahara Satoru , Yokoyama Shigeyuki , Takaku Hiroshi
Published: July 2002

Analogues of the homopyrimidine oligonucleotide dT15 that contained phosphorothioate bonds of a mixture of diastereoisomers or one of the two stereoisomers (either Rp or Sp) were synthesized. The analogues were mixed under conditions conducive to the formation of triple‐stranded assemblies. The mixtures were characterized by their thermal stabilities (T
m values), CD spectra, and gel electrophoresis pattern. The 34‐mer duplexes containing 15 central purines on one strand and 15 complementary pyrimidines on the other strand gave no detectable triple helix upon combination with dT15S14. On the other hand, 34‐bp duplexes with dT15S1, having Rp or Sp, formed triple helixes. This suggests that a steric factor plays an important role in triple helix formation.
Identification of a key domain in annexin and 14‐3‐3 proteins that stimulate calcium‐dependent exocytosis in permeabilized adrenal chromaffin cells
Authors: Roth Dagmar , Morgan Alan , Burgoyne Robert D.
Published: July 2002

Calcium‐dependent secretion in digitonin‐permeabilized adrenal chromaffin cells is stimulated by exogenous annexin II and 14‐3‐3 proteins. These proteins share a conserved domain that has been suggested to be involved in specific protein‐protein interactions. We examined whether this domain was involved in secretion by using a synthetic peptide (P16) of sequence KGDYQKALLYLCGGDD corresponding to the C‐terminus of annexin II. P16, but not truncated peptides, prevented the stimulation of secretion by 14‐3‐3 proteins and produced a partial inhibition of control secretion. These data suggest that the shared annexin/14‐3‐3 domain is important in the mechanisms controlling Ca2+‐dependent secretion and may play a key role in protein‐protein interactions during exocytosis.
A commentary on: ‘Unexpected intron location in non‐vertebrate globin genes’, by: Moens et al. (FEBS Letters, 312 (1992) 105‐109) (FEBS 12294)
Authors: Pohajdak Bill , Dixon Brian
Published: July 2002

We are submitting this commentary in order to prevent the confusion which Moens et al. may provoke in the minds of readers. Our comments are intended to point out the exclusion of some major issues and some errors which may mislead readers of this paper.
Neopterin modulates toxicity mediated by reactive oxygen and chloride species
Authors: Weiss Günter , Fuchs Dietmar , Hausen Arno , Reibnegger Gilbert , Werner Ernst R. , Werner-Felmayer Gabriele , Semenitz Erich , Dierich Manfred P. , Wachter Helmut
Published: July 2002

Neopterin, a pyrazino‐pyrimidine derivative, is synthesized in excess by human monocytes/macrophages upon stimulation with interferon‐γ, a cytokine derived from activated T‐cells. Neopterin is furthermore produced constitutively. A relatively constant ratio between neopterin and its reduced form, 7,8‐dihydroneopterin, has been described in human serum. In the study presented here we tested the ability of neopterin and its reduced form to modulate the effects of cytotoxic substances like hydrogen peroxide or hypochlorous acid and N‐chloramine derivatives. We show that 7,8‐dihydroneopterin potently reduces biological and chemical effects of these substances independently from the pH value. In contrast, at slightly alkaline pH (pH 7.5) neopterin enhances hydrogen peroxide and chloramine‐T activity. This is demonstrated by increase of signal intensity in a luminol assay and also by enhancement of toxicity towards bacteria. Thus, the macrophage derived substance neopterin is able both to enhance and to reduce cytotoxicity in dependence of pH value and its oxidation state, and it may have a pivotal role in modulation of macrophage mediated effector mechanism.
VAT‐1 from Torpedo is a membranous homologue of zeta crystallin
Authors: Linial Michael , Levius Orit
Published: July 2002

VAT‐1 is a major protein from Torpedo synaptic vesicles. A protein data‐base search revealed a striking homology to ξ crystallin from guinea pig lens. The overall amino‐acid identity is 27%, and 58% similarity is reached by including conserved substitutions. The highest similarity (60% to 85%) between the two proteins is observed in five discrete domains, which are also conserved in zinc‐dependent dehydrogenases, particularly in the alcohol dehydrogenase family. The cofactor‐binding domain of oxidoreductases is conserved in VAT‐1 and in ξ crystallin. VAT‐1 preferably binds NADPH in the presence of zinc. In contrast with its homologous proteins, VAT‐1 is an integral membrane protein of synaptic vesicles.