bluuebunny/crossref_metadata_2025 huggingface dataset:
Disulfide bridges in human complement component C3b
Authors: Dolmer Klavs , Sottrup-Jensen Lars
Published: July 2002

The disulfide bridges of human complement component C3b, derived from C3 by removal of the 77‐residue C3a, have been determined. The 10 bridges are Cys537‐Cys794, Cys605‐Cys640, Cys851‐Cys1491, Cys1079‐Cys1169, Cys1336‐Cys1467, Cys1367‐Cys1436, Cys1448‐Cys1489, Cys1496‐Cys1568, Cys1515‐Cys1639, and Cys1615‐Cys1624. Including the 3 bridges in C3a (Cys670‐Cys698, Cys672‐Cys705, and Cys685‐Cys706) previously determined by high‐resolution X‐ray crystallography [Hoppe‐Seyler's Z. Physiol. Chem. 361 (1980) 1389‐1399] all disulfide bridges of C3 are localized. C3 and the strongly related C4 and C5 are members of the α2‐macroglobulin superfamily. The predicted bridge patterns of C4 and C5 are discussed and compared with that of α2‐macroglobulin.
Conformational changes in subdomain‐2 of G‐actin upon polymerization into F‐actin and upon binding myosin subfragment‐1
Authors: Fievez Stéphane , Carlier Marie-France
Published: July 2002

The susceptibility of subdomain‐2 of actin to different proteases has been examined, for G‐actin, F‐actin, G‐actin‐S1(A2) and F‐actin‐S1(A2) complexes on a comparative basis. The sites of subtilisin, α‐chymotrypsin and trypsin attack, exposed on G‐actin, are protected in F‐actin, F‐actin‐S1(A2) as well as in the G‐actin‐S1(A2) complex. In contrast, a new cleavage site (Arg39‐His40) for ArgC protease, which is protected in G‐actin, is exposed in G‐actin‐S1(A2) as well as in F‐actin and F‐actin‐S1(A2). These results are consistent with the previously proposed structural analogy between the ternary (G‐actin)2S1 and the F‐actin‐S1 complexes, and provide information on the mechanism of S1‐induced polymerization of G‐actin.
Adenosine analogs inhibit the guanine‐7‐methylation of mRNA cap structures
Authors: Oxenrider Keith A. , Bu Guojun , Sitz Thomas O.
Published: July 2002

The adenosine analogs neplanocin A and deazaneplanocin A were observed to inhibit the in vivo guanine‐7‐methylation of mRNA cap structure using a new assay for hypomethylated RNA. Treatment of cultured mammalian cells with these adenosine analogs resulted in the same extent of hypomethylation of cap structure as did ethionine injection in mice. Neplanocin A and its non‐metabolizable analog 3‐deazaneplanocin A show the same maximal level of inhibition of methylation suggesting that these adenosine analogs exert their effects by elevating S‐adenosylhomocysteine levels rather than by conversion to other inhibitory compounds.
Inhibition of myosin ATPase activity by human myasthenia gravis antibodies reactive with the acetylcholine receptor
Authors: Mohan Sumathy , Krolick Keith A.
Published: July 2002

Antibodies, obtained from myasthenia gravis patients, with reactivity for an immunodominant region of the nieotinic acetylcholine receptor were found to also react with muscle myosin. Since amino acid sequence analyses have previously suggested possible serological relationships between AChR and a head region sequence of myosin heavy chain, cross‐reactive antibodies were examined for their ability to interfere with ATPase activities associated with this region of myosin. Results indicated that AChR‐specific antibodies purified from MG patient serum by binding to and elution from antigen columns were found to inhibit Ca2+‐dependent, myosin‐associated ATPase activity; interestingly, this inhibition appeared to be relatively selective in that neither K+(EDTA)‐dependent nor Mg2+‐dependent ATPase activities were sensitive to antibody‐mediated interference.
Opposite regulations by androgenic and thyroid hormones of V1 myosin expression in the two types of rabbit striated muscle: skeletal and cardiac
Authors: d'Albis Anne , Couteaux René , Janmot Chantal , Mira Jean-Claude
Published: July 2002

The finding that VI cardiac myosin is expressed in masticatory skeletal muscles of the rabbit [1] provided a unique opportunity for comparing the hormonal regulation of VI in skeletal and cardiac muscles. Thyroid hormones had no significant effect on the postnatal expression of VI in masticatory muscles, but increased this expression in cardiac ventricles. In contrast, androgenic hormones reduced VI expression in masticatory muscles, but did not affect it significantly in cardiac ventricles. Modulation of VI gene transcription in striated muscle is thus shown here to depend both on the target muscle and on the hormone.
Prediction of a novel topology in the N‐terminal, 14 kDa fragment of ada protein
Authors: Swindells Mark B.
Published: July 2002

Previously determined protein structures have been analysed, in order to find folding motifs similar to that proposed by NMR spectroscopy, for the N‐terminal, 14 kDa fragment of the Ada protein. The analyses reveal only limited similarities with the NMR‐derived structural data and strongly suggest that this region of the Ada protein adopts a previously unobserved topology. Characteristic structural features, which arise from the inferred chain connectivity, are examined through comparisons with other structures. Using this information, the topology of the Ada protein 14 kDa fragment has been predicted in order to provide structural data not yet attainable from NMR experiments.
Saccharomyces cerevisiae multifunctional protein RAP1 binds to a conserved sequence in the Polyoma virus enhancer and is responsible for its transcriptional activity in yeast cells
Authors: Pollice Alessandra , Ciaramella Maria , Pulitzer John F.
Published: July 2002

The Polyoma virus enhancer (A + B domain) activates transcription in Saccharomyces cerevisiae when joined to appropriate yeast promoter elements. We demonstrate by DNase I footprints and inhibition of binding by specific antibody, that the yeast protein RAP1 binds to the B‐domain of the Polyoma enhancer and, at least in some promoter contexts, is responsible for transcriptional activity of the enhancer B‐domain in yeast. Close matches to a consensus RAP1‐binding site are also present in other viral enhancers.
E. coli expression and characterization of a mutant troponin I with the three cysteine residues substituted
Authors: Kluwe Lan , Maeda Kayo , Maéda Yuichiro
Published: July 2002

A TnI cDNA was cloned from rabbit fast skeletal muscle, and site‐directed mutagenesis was applied to replace all the three cysteine residues, Cys‐48 and Cys‐64 by Ala and Cys‐133 by Ser. The mutant and wild‐type TnI were expressed in E. coli and purified to homogeneity. No significant functional differences were observed between the mutant and the authentic TnI in terms of the interactions with TnT and TnC, and the ability of the reassembled Tn complex to regulate the acto‐S1 ATPase activity in a calcium‐dependent manner. These findings suggest that none of the cysteine residues in TnI are essential for the function of this protein and can be replaced to obtain a non‐oxidizable mutant TnI which is much easier to handle and suitable as an alternative to the authentic TnI for various purposes, such as crystallization of TnI and the whole Tn, and 1H NMR studies.
Enrichment of proteinase activity in deteriosomes, a new class of microvesicles
Authors: Yao Kening , Thompson John E.
Published: July 2002

Non‐sedimentable microvesicles, termed deteriosomes, have been isolated from the cytosol of bean cotyledons by ultrafiltration, and found to be enzymatically active. Specifically, they possess proteinase activity that is able to catabolize exogenous proteins as well as deteriosome proteins. The proteolytic activity is inhibited by heat‐denaturation and known proteinase inhibitors (iodoacetate and phenylmethylsulfonyl fluoride), and the pattern of deteriosome‐associated proteinase activity changes with advancing senescence of the cotyledon tissue. Deteriosomes are formed by blebbing from membranes. The finding that they possess proteinase activity is consistent with an earlier proposal [(1991) Proc. Natl. Acad. Sci., USA 88, 2269‐2273] that they are involved in membrane turnover and serve as a vehicle for moving bilayer‐destabilizing phospholipid and protein catabolites out of membranes into the cytosol for further processing. The data also indicate that a significant proportion of the proteinase activity traditionally considered to be cytosolic is associated with deteriosomes.
Random‐splitting of tRNA transcripts as an approach for studying tRNA‐protein interactions
Authors: Aphasizhev Ruslan , Beresten Sergey , Pugachev Vladimir , Kisselev Lev
Published: July 2002

Location of phosphodiester bonds essential for aminoacylation of bovine tRNTrp was identified using a randomly cleaved transcript synthesized in vitro. It was found that cleavage of phosphodiester bonds after nucleotides in positions 21, 22, 36–38, 57–59, 62 and 64 were critical for aminoacylation capacity of tRNATrp‐transcript. These cleavage sites were located in the regions of tRNA molecule protected by the cognate synthetase against chemical modification and in the regions presumably outside the contact area as well. These results indicate that for maintenance of aminoacylation ability the intactness of the certain regions of the tRNA backbone structure is necessary. Random splitting of non‐modified RNA with alkali followed by separation of active and inactive molecules and identification of cleavage sites developed in this work may become a general approach for studying the role of RNA covalent structure in its interaction with proteins.
Regulation of expression of β‐galactoside α2, 6‐sialyltransferase in a rat tumor, Zajdela ascitic hepatoma
Authors: Jain Neeraj , Sudhakar Ch. , Das M.R.
Published: July 2002

Tumor cell surface sialic acid levels determine a number of important properties governing cellular interactions and cell‐cell communication. Towards understanding the mechanism of regulation of sialic acid levels upon cellular transformation, we have studied the regulation of expression of β‐galactoside α2, 6‐sialyltransferase in a rat tumor, the Zajdela ascitic hepatoma. We demonstrate distinct differences in the regulation of expression of the enzyme in the tumor cells as compared to normal liver cells. The expression of sialyltransferase is regulated both at the transcriptional and post‐transcriptional level in a tissue‐specific manner.
Irsogladine is a potent inhibitor of angiogenesis
Authors: Sato Yasufumi , Morimoto Akio , Kiue Akira , Okamura Kazuki , Hamanaka Ryoji , Kohno Kimitoshi , Kuwano Michihiko , Sakata Toshiie
Published: July 2002

We describe a novel inhibitor of angiogenesis, Irsogladine, an anti‐ulcer drug. Irsogladine inhibited plasminogen activator synthesis of, and tube formation by, human microvascular endothelial cells in type 1 collagen gel treated with an angiogenic growth factor, EGF. Furthermore, Irsogladine administered orally significantly inhibited in vivo angiogenesis in mice. Irsogladine may be useful in the treatment of diseases associated with angiogenesis.
Purification and characterization of the cystinyl bond cleaving yeast aminopeptidase yscXVI
Authors: Tisljar Ursula , Wolf Dieter H.
Published: July 2002

Aminopeptidase yscXVI was purified from the yeast Saccharomyces cerevisiae. By SDS‐PAGE the enzyme has a molecular weight of 45,000 Da, and in chromatofocusing, elution was observed at pH 6.2. The synthetic substrate cystinyl‐4‐nitroanilide (K
m 22.5μM, V
max 12.9 mU/mg) is cleaved most efficiently in the pH range 7–8. Besides cleaving this standard substrate, aminopeptidase yscXVI acts on several other 4‐nitroanilide substrates with unsubstituted N‐terminal l‐amino acids. Highest hydrolysis rate was measured with Lys‐4‐nitroanilide and Leu‐4‐nitroanilide. The activity of aminopeptidase yscXVI is abolished by chelating agents and restored by Zn2+, Mn2+ and Co2+ ions. Bestatin and amastatin are both strong inhibitors of the enzyme, with K
i values of 0.53μM and 0.93μM, respectively. Aminopeptidase yscXVI is detectable in the logarithmic growth phase, stationary phase, and in starved cultures of yeast.
Region‐specific expression of multiple lactase‐phlorizin hydrolase genes in intestine of rabbit
Authors: Villa Marco , Brunschwiler Django , Gächter Thomas , Boll Werner , Semenza Giorgio , Mantei Ned
Published: July 2002

We have identified a total of 4 sequences coding for lactase‐phlorizin hydrolase (LPH) in the rabbit. Individual rabbits each yielded three different LPH cDNA sequences, or three chromosomal segments containing exon 1 of the LPH gene, representing either three genes or allelic variants of two genes. The three sequences were found in differing proportions in intestinal mRNA depending on the position along the small intestine from which the RNA was isolated. If all these mRNAs are translated, posttranslational mechanisms previously implicated in the regulation of LPH in the rabbit will be acting on different enzyme species in different parts of the intestine. However, we find no evidence for more than one LPH gene in the rat, and have previously shown that humans have only one LPH gene. p]Lactase‐phlorizin hydrolase; Developmental regulation; Small intestine; Gene family
Function of saposin C in the reconstitution of glucosylceramidase by phosphatidylserine liposomes
Authors: Vaccaro Anna Maria , Tatti Massimo , Ciaffoni Fiorella , Salvioli Rosa , Maras Bruno , Barca Alessandra
Published: July 2002

The function of saposin C (Sap C), a glucosylceramidase activator protein, in the enzyme stimulation by phosphatidylserine (PS) liposomes has been investigated. Using gel filtration experiments evidence was obtained for Sap C binding to PS large unilamellar vesicles (LUV) but not to glucosylceramidase. PS LUV, which by themselves are unable to tightly bind and stimulate the enzyme, acquire the capacity to also bind the enzyme after interaction with Sap C, making it express its full activity. Our results indicate that the primary step in the Sap C mode of action resides in its association with PS membranes; in turn, this association promotes the interaction between the membranes and glucosylceramidase.
Phospholipase D treatment enhances gonadotrophin receptor‐coupled adenylate cyclase activity in isolated bovine luteal cells
Authors: Budnik Lygia T. , Mukhopadhyay Amal K.
Published: July 2002

LH‐stimulated adenylate cyclase activity in membrane preparations of bovine luteal cells could be enhanced by treating the cells with either phospholipase D or its hydrolysis product, phosphatidic acid. Similar augmentary effects were also produced following treatment of the cells with EGF. Moreover, EGF could stimulate the formation of [3H]phosphatidic acid in [3H]myristic acid preloaded cells, suggesting that EGF is able to activate cellular phospholipase D. Also, PMA was able to increase the phosphatidic acid formation with a parallel increase in the adenylate cyclase activity. We propose, therefore, that phosphatidic acid may act as an intracellular second messenger linking EGF‐mediated activation of phospholipase D with the sensitization of LH receptor‐coupled adenylate cyclase signalling system.
Nucleic acid‐binding regions of the second‐largest subunit of Drosophila RNA polymerase II identified by Southwestern blotting
Authors: Kontermann Roland E. , Bautz Ekkehard K.F.
Published: July 2002

Analysing overlapping bacterially expressed fragments of the second‐largest subunit of Drosophila melanogaster RNA polymerase II in Southwestern DNA binding assays we have identified regions that have the potential to bind nucleic acids non‐specifically. A region exhibiting strong DNA binding is located in the N‐terminal part of the molecule (amino acids 357–504) and some weak DNA binding is observed for the C‐terminal part (amino acids 860–1160). The non‐specific DNA binding behavior of these regions is similar to that of the native enzyme. Most of the known mutations responsible for rifampicin resistance map to a region of the Escherichia coli β subunit corresponding to the N‐terminal nucleic acid‐binding region, indirectly supporting the notion that this region participates in interaction with the RNA transcript in ternary complexes.
Intergenic suppression in a β subunit mutant with defective assembly in Escherichia coli F1ATPase
Authors: Miki Junji , Tsugumi Sadamu , Ikeda Hiromi , Kanazawa Hiroshi
Published: July 2002

Substitution of Leu‐40 by Pro in the β subunit (βL40P) of Escherichia coli F1‐ATPase caused a decrease in the amount of the α and β subunits on the membranes. A revertant strain, Re50, carrying no suppression mutations in the uncD gene encoding the β subunit, was isolated from the βL40P mutant. The uncA gene from this revertant was amplified by PCR, and cloned into an expression plasmid. The expression plasmid carrying the uncA gene from the revertant was used for genetic suppression assays. The suppression mutation in Re50 was in the α subunit, and it recovered the assembly of the α and β subunits into the F1F0 complex and the ATPase activity to 50% that of the wild type. In Re50, Leu‐111 was substituted by Gln in the α subunit. These results suggest that the regions including Leu‐40 in the β subunit and Leu‐111 in the α subunit are located close together and interact with each other, either directly or indirectly.
X‐band ESEEM spectroscopy of 15N substituted native and inhibitor‐bound superoxide dismutase
Authors: Dikanov Sergei , Felli Isabella , Viezzoli Maria-Silvia , Spoyalov Andrei , Hüttermann Jürgen
Published: July 2002

The hyperfine couplings of the remote nitrogens of histidine ligands are determined for the first time by an X‐band ESEEM spectroscopy study of 15N‐substituted superoxide dismutase (SOD). They show a significant difference between two groups of ligands with different orientation relative to the metal ion. The ESEEM spectra of 15N SOD with cyanide as an inhibitor containing 14N and 15N are also discussed. They allow some conclusions to be drawn about structural changes upon inhibitor binding and indicate the necessity of further multifrequency investigations.
CT‐Boxes are involved in control of the rat insulin II gene expression
Authors: Rommel Christian , Leibiger Ingo B. , Leibiger Barbara , Walther Reinhard
Published: July 2002

Expression of the rat insulin II gene is controlled mainly at the level of transcription initiation by multiple factors binding to specific cis‐acting DNA‐elements in the regulatory region. We have shown that two elements (CT‐motifs) located between nucleotides −83 and −76 (CT‐1) and −204 and −197 (CT‐2) are involved in transcriptional regulation in the insulin‐producing cell line HIT M2.2.2. Transient expression analysis of 5'‐deletion as well as block replacement mutants revealed that CT‐1 and CT‐2 are mutational sensitive. Gel mobility shift assays showed that both motifs bind similar nuclear factors. Our results suggest the involvement of a third CT‐motif located directly upstream of CT‐2 on the complementary strand.
Binding of GM1‐ganglioside to a synthetic peptide derived from the lysosomal sphingolipid‐activator‐protein saposin B
Authors: Champagne Marie-Josée , Lamontagne Sonia , Potier Michel
Published: July 2002

Saposin B is a lysosomal sphingolipid‐activator‐protein which activates GM1‐ganglioside hydrolysis by lysosomal β‐galactosidase. To identify the structural elements of saposin B implicated in sphingolipid binding, we studied a synthetic peptide corresponding to a predicted α‐helix, sapB‐18, spanning residues 52 to 69 of saposin B. The circular dichroism spectrum of sapB‐18 at pH 4.4 was consistent with a 44% α‐helix content. As shown by intrinsic Tyr fluorescence studies of sapB‐18, this peptide binds the GM 1 ‐ganglioside with a K
d of about 7μM. Thus, we suggest that a putative amphipathic α‐helix between residues 52 and 69 of saposin B plays a major role in the recognition and binding of GM1‐ganglioside by saposin B.
Making a small enzyme smaller; removing the conserved loop structure of hen lysozyme
Authors: Pickersgill Richard , Varvill Katherine , Jones Sean , Perry Barry , Fischer Bernhard , Henderson Ian , Garrard Sarah , Sumner Ian , Goodenough Peter
Published: July 2002

Engineering a smaller lysozyme is a challenge for both random and site‐directed mutagenesis. This paper illustrates the power of knowledge‐based protein engineering in the design of a smaller lysozyme that folds correctly and has activity against bacterial cell walls. In this smaller lysozyme the conserved disulphide bridged loop is replaced by a short loop. The long loop was selected because it buries a predominantly hydrophilic surface. The short loop was discovered by searching for appropriate fragments in the protein databank. This approach is important in the design of small enzymes useful to the food industry.
Purification, crystallization and preliminary X‐ray diffraction analysis of recombinant human neutrophil‐activating peptide 2 (rhNAP‐2)
Authors: Kungl Andreas J. , Machius Mischa , Huber Robert , Schwer Christine , Lam Charles , Aschauer Heinz , Ehn Gerald , Lindley Ivan J.D. , Auer Manfred
Published: July 2002

The potent activator and chemoattractant for human neutrophils, neutrophil‐activating peptide 2 (NAP‐2), has been cloned and expressed in Escherichia coli. The protein has been purified to homogeneity (> 98%) by a series of chromatographic techniques, including reversed phase HPLC. The biological activity of recombinant human NAP‐2 (rhNAP‐2), characterized by the induction of elastase release from human neutrophils, was found to be comparable to natural NAP‐2. rhNAP‐2 has been crystallized by the hanging drop vapor diffusion method. The crystals belong to space group P222 with unit cell dimensions of a = 30.8 Å, b = 39.5 Å and c = 95.3 Å. A packing density of 3.8 Å3/Da with a solvent content of approximately 68% is obtained when one molecule per asymmetric unit is assumed. The crystals were shown to diffract to beyond 2.0 Å on a conventional X‐ray source. They are stable to X‐rays for several days and are thus suitable for high resolution structure determination.
Isolation of tyrosine kinase related genes expressed in the early hematopoietic system
Authors: Larsson-Blomberg Lena , Dzierzak Elaine
Published: July 2002

Transmembrane tyrosine kinase receptors are involved in cellular interactions which promote proliferation and differentiation of many cell types. To identify receptor tyrosine kinases important in embryonic hematopoietic cell development we have utilized the polymerase chain reaction (PCR) and degenerate oligonucleotides for isolation of such genes from mouse yolk sac and fetal liver. Sequence analysis of PCR amplified cDNAs from these hematopoietic sites of day 8 and 14 embryos, resulted in the isolation of nine tyrosine kinase and three serine/threonine kinase related clones. Two of these receptors, tek and flk‐1, are expressed in both yolk sac and fetal liver and have been shown previously to be important for endothelial cell development. Two other clones, 9B4 and 9A2 appeared novel upon isolation but have been recently described as ryk and SK2 (rat homologue). Here we describe the twelve isolated kinases, the specific expression patterns of flk‐1, tek and ryk kinases and their potential relationship to the development of the hematopoietic system.
Priming effect of hydroxyapatite on the chemiluminescence response in human polymorphonuclear leukocytes
Authors: Nagase Mitsuo , Nishiya Hajime , Noda Masatoshi
Published: July 2002

In order to determine whether hydroxyapatite modulates the response of polymorphonuclear leukocyte (PMN) to oxidative stimuli, human PMNs were incubated with a non‐activating concentration (1 or 10 μg/ml) of hydroxyapatite prior to stimulation with N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP; 0.1 or 1 μM), phorbol 12‐myristate 13‐acetate (PMA; 100 pg/ml), sodium fluoride (50 μM), zymosan (1 μg/ml), or the calcium ionophore A23187 (0.1 μM). Chemiluminescence was measured with an automatic microcomputer‐controlled luminescence analyzer at 37°C. Hydroxyapatite alone did not stimulate chemiluminescence at concentrations below 10 μg/ml. Levels 300–400% higher than ‘stimulus only’ controls without preincubation with hydroxyapatite have been recorded. This synergism between hydroxyapatite and subsequent stimuli reveals a new activity of hydroxyapatite and suggests that particulate material may prepare PMNs for an exaggerated inflammatory response to other phlogistic mediators. This is the first report demonstrating PMNs primed with particulate material.
Transcription initiation sites for the potato mitochondrial gene coding for subunit 9 of ATP synthase (atp9)
Authors: Lizama Luis , Holuigue Loreto , Jordana Xavier
Published: July 2002

The potato mitochondrial atp9 gene has a simple expression pattern. To determine where transcription initiates, primary mitochondrial RNAs were labeled by in vitro capping and hybridized to the 5′ flanking sequences of the atp9 gene. A single transcription initiation region was identified. Primer extension and nuclease S1 protection analyses were used to precisely map the transcript 5′ termini in this region. These results indicate that transcription initiates at 121–128 bp upstream of the atp9 open reading frame, in a sequence which does not present any homology with proposed consensus sequences for plant mitochondrial promoters. Nuclease S1 protection were also used to map 3′ termini 67–71 nucleotides downstream of a putative single‐stem loop structure.
Altered chemotaxis of a Bacillus sphaericusl‐ethionine‐resistant sporulation mutant a probable link between chemotaxis and sporulation
Authors: Andreev J. , Dibrov P.A. , Braun S.
Published: July 2002

A UV irradiation‐induced mutant of B. sphaericus 2362 whose sporulation was inhibited neither by natural amino acids nor by l‐ethionine was selected. The mutant (A61) grew slowly in rich amino acid medium and contained increased concentrations of heat‐resistant spores throughout the growth. Slow growth of A61 was related to continuous presence of aging and sporulating cells even when the medium was rich in nutrients. The ability of the mutant to sense nutrient presence in the environment and to relate this information to systems regulating the switch from vegetative growth to sporulation seem to be damaged. A61 also demonstrated impaired chemotaxis. In contrast to the parent strain, only few amino acids elicited chemotactic response in A61. Methylation of the A61 methyl‐accepting chemotaxis protein(s) was lower than that of the parent strain by one order of magnitude. Spontaneous fast‐growing phenotypic revertants of A61 displayed sporulation behavior characteristic of B. sphaericus 2362. Their chemotaxis to amino acids was considerably improved. To some amino acids, it proved to be even stronger than in the original strain, B. sphaericus 2362. It is suggested, that methyl transfer events originating in the chemotactic system are involved in the triggering of sporulation, the A61 mutation being located in this signalling pathway.
Binding of GM1 ganglioside to a synthetic peptide derived from the lysosomal sphingolipid activator protein saposin B
Authors: Champagne Marie-Josée , Lamontagne Sonia , Potier Michel
Published: July 2002

Saposin B is a lysosomal sphingolipid activator protein which activates GM1 ganglioside hydrolysis by lysosomal β‐galactosidase. To identify the structural elements of saposin B implicated in sphingolipid binding, we studied a synthetic peptide corresponding to a predicted α‐helix, sapB‐18, spanning residues 52–69 of saposin B. The circular dichroism spectrum of sapB‐18 at pH 4.4 was consistent with a 44% α‐helix content. As shown by intrinsic Tyr fluorescence studies of sapB‐18, this peptide binds the GM1 ganglioside with a K
d of about 7 μM. Thus, we suggest that a putative amphipathic α‐helix between residues 52 and 69 of saposin B plays a major role in the recognition and binding of GM1 ganglioside by saposin B.
Metabolic pathways in Ehrlich ascites tumor cells recovering from a low bioenergetic status
Authors: Dell'Antone Paolo
Published: July 2002

Ehrlich ascites tumor cells were found to be in a low bioenergetic status, as evaluated by acridine orange uptake and ATP content, when resuspended in a glucose medium shortly after removal from the animal. Dye uptake as well as ATP content then increased for about 2 h at room temperature. This effect was only slightly inhibited by oligomycin. Cells resuspended in a glucose‐free medium initially showed high dye uptake and ATP level, which were stable over time: in this case oligomycin caused a drop in both dye uptake and ATP level. The above findings, which are indicative of a marked Crabtree effect in Ehrlich ascites tumor cells, means that it is unlikely that limiting ADP and Pi play an important role in the glucose‐induced inhibition of oxidative phosphorylation in this system.
Electron transfer and ligand binding in terminal oxidases Impact of recent structural information
Authors: Brunori M. , Antonini G. , Giuffre A. , Malatesta F. , Nicoletti F. , Sarti P. , Wilson M.T.
Published: July 2002

A consensus structure for the active site of terminal oxidases has been recently proposed by Hosler et al. [(1993) J. Bioenerg. Biomem. 25, 121–1351]. We exploit the novel structural information to propose a hypothesis for the large difference in the rate of internal electron transfer found when experiments are started either with the reduced or with the oxidized enzyme. This rationale also allows us to discuss the oxidation state of the prevailing oxygen reacting species with reference to the concentration of the two substrates (oxygen and cytochrome c) and to the structural state of the oxidase.
The first hydrophobic segment of the ABC‐transporter, Ste6, functions as a signal sequence
Authors: Kölling Ralf , Hollenberg Cornelis P.
Published: July 2002

The Ste6 protein of Saccharomyces cerevisiae is a member of the ABC‐transporter family containing 12 putative membrane spanning segments. To test whether Ste6 is inserted into the endoplasmic reticulum (ER) membrane by a sequential insertion mechanism we constructed a Ste6‐invertase fusion containing the first hydrophobic segment of Ste6 fused to invertase lacking its own signal sequence. The resulting protein became glycosylated demonstrating that it was translocated across the ER‐membrane. The finding that the N‐terminal hydrophobic segment of Ste6 is recognized by the ER‐translocation machinery suggests that Ste6 is inserted sequentially into the ER‐membrane. Furthermore, our experiments support the Nin orientation of Ste6 predicted from the Ste6 sequence. Several findings suggest that invertase is cleaved from the Ste6 membrane anchor: (i) the gel mobility of deglycosylated wild‐type invertase and fusion protein derived invertase is the same; (ii) the periplasmic invertase activity is found in the cell wall fraction, i.e. it is not associated with the cell body; (iii) a signal peptide cleavage site is predicted in the Ste6 sequence. Although the membrane anchor appeared to be cleaved, most of the invertase was retained in the ER, probably due to aggregate formation.
Oxygen‐derived radicals stimulate renin release of isolated juxtaglomerular cells
Authors: Galle Jan , Herzog Christel , Schollmeyer Peter , Wanner Christoph
Published: July 2002

We assessed effects of reactive oxygen metabolites on renin release of juxtaglomerular cells (JG‐cells) prepared in primary culture from mouse kidneys. Renin activity was measured in culture supernatants and cells. Basal renin release was increased by incubation of JG‐cells with xanthine/xanthine oxidase from 26 ± 1% up to 58 ± 3% of total activity. This increase was slightly inhibited by superoxide dismutase, and was eliminated by addition of catalase, implicating H2O2 as an intermediate product in the stimulatory cascade. H2O2 applied exogeneously dose‐dependently stimulated renin release up to 55 ± 2%; this effect was also prevented by catalase. We propose that reactive oxygen metabolites stimulate renin release in isolated JG‐cells. This could have important implications in inflammatory kidney diseases.
Production of double‐stranded RNA during synthesis of bromouracil‐substituted RNA by transcription with T7 RNA polymerase
Authors: Cao Tuong Luan , Revers Frédéric , Cazenave Christian
Published: July 2002

Using T7 RNA polymerase we synthesized a short oligoribonucleotide containing bromouracil by in vitro transcription of a synthetic DNA template. Whereas the major transcript obtained had the expected size and was apparently homogeneous on a sequencing gel, additional analysis revealed the presence of double‐stranded RNA in this preparation. As this was not observed when the same template was transcribed in the presence of uracil, we hypothesize that bromouracil promoted the apparition of double‐stranded ‘parasitic’ RNA presumably by favouring priming for the RNA‐dependent RNA synthesis of the T7 RNA polymerase or by facilitating an end‐to‐end copy mechanism.
Functional interaction between the epidermal growth factor receptor and c‐Src kinase activity
Authors: Oude Weernink Paschal A. , Ottenhoff-Kalff Astrid E. , Vendrig Marijke P. , van Beurden Ellen A.C.M. , Staal Gerard E.J. , Rijksen Gert
Published: July 2002

To study the relationship between the tyrosine kinase c‐Src and the epidermal growth factor receptor (EGF‐R), we used the breast cancer cell line ZR75‐1, which was transfected with the EGF‐R. The EGF‐R transfected cell line expressed 60 times more EGF‐R than a control cell line transfected with the empty vector. In the presence of EGF, the EGF‐R over‐expressing cell line grew much faster than the control cell line. Both cell lines expressed approximately equal amounts of c‐Src. However, the cell line over‐expressing the EGF‐R showed a twofold enhancement of c‐Src kinase activity after EGF stimulation. The activation of c‐Src kinase by EGF was confirmed in other EGF‐R expressing cell types.
The immediate vicinity of mouse metallothionein‐I gene contains two sites conferring glucocorticoid inducibility to the heterologous promoter
Authors: Plisov Sergey Y. , Nichiporenko Marina G. , Shkapenko Alla L. , Kumarev Victor P. , Baranova Ludmila V. , Merkulova Tatyana I.
Published: July 2002

Glucocorticoid responsive elements (GREs) located −252 to −209 bp upstream and +1011 to +1054 bp downstream of the transcription initiation site of the mouse metallotionein‐I (mMT‐I) gene were identified in transient transfection eperiments. However, the promoter region of the mMT‐I gene (−330 to +70 bp) was found to provide low, if any, glucocorticoid induction of the linked CAT gene, while showing strong cadmium regulation, comparable with the in vivo level.
The l‐histidine‐mediated enhancement of hydrogen peroxide‐induced cytotoxicity is a general response in cultured mammalian cell lines and is always associated with the formation of DNA double strand breaks
Authors: Cantoni Orazio , Sestili Piero , Brandi Giorgio , Cattabeni Flaminio
Published: July 2002

Micromolar concentrations of l‐histidine increase the cytotoxicity of hydrogen peroxide in a number of cell lines including CHO(hamster), EAHY, McCoy's, U937 and CCRF‐CEM (human), Vero (monkey) and SC‐1 (mouse). Importantly, these cell lines displayed differentt degrees of sensitivity to H2O2 alone and the extent of enhancement elicited by the amino acid was more pronounced in resistant cell lines. The increased cytotoxicity was invariably associated with the formation of DNA DSBs and a remarkable correlation was found by plotting the level of DNA DSBs against the cytotoxic response. These results strongly support the hypothesis that the mechanism whereby l‐histidine increases the toxicity elicited by H2O2 involves the formation of DNA DSBs and are consistent with the possibility that the amino acid might participate in the regulation of the physio‐pathological response to oxidative stress in mammals.
Growth factors stimulate phosphorylation of CTP:Phosphocholine cytidylyltransferase in HeLa cells
Authors: Wieprecht Marcus , Wieder Thomas , Geilen Christoph C. , Orfanos Constantin E.
Published: July 2002

The effect of insulin and epidermal growth factor on the phosphorylation of CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) was investigated in HeLa cells. For the first time, cytidylyltransferase phosphorylation was shown to be influenced by growth factors in cell culture experiments. The rephosphorylation of cytidylyltransferase after an oleate‐mediated dephosphorylation and translocation to membranes was increased after 2 min in the presence of insulin or epidermal growth factor by 99% and 76%, respectively, compared with controls. However, the increased phosphorylation of cytidylyltransferase did not have an effect on its subcellular distribution. Furthermore, purified cytidylyltransferase preincubated with alkaline phosphatase is a substrate for p44mapk, a member of the mitogen‐activated protein (MAP) kinase family downstream of the growth factor receptors, in vitro. In accordance with the in vivo data, in vitro phosphorylation of cytidylyltransferase by p44mapk occurred after 2 min.
Inactivation of Acacia confusa trypsin inhibitor by site‐specific mutagenesis
Authors: Hung Chih-Hung , Lee Ming-Chih , Lin Jung-Yaw
Published: July 2002

Native Acacia confusa trypsin inhibitor (ACTI) contains two disulphide bonds; one is an intrachain disulphide bond (Cys40‐Cys86), located in the A‐chain, while the other is an interchain disulphide bond (Cys133‐Cys141) connecting the A‐ and B‐chain; the inhibitor consists of 175 amino acid residues, 136 residues in the A‐chain and 39 residues in the B‐chain. The putative reactive site of ACTI is located at Lys64, while for all other Kunitz family trypsin inhibitors it is at Arg64. When the Lys64 residue of ACTI was converted into Ile or Arg by site‐specific mutagenesis, the K64I mutant completely lost its inhibitory activity but the K64R mutant retained most of its inhibitory activity. The C133G mutant lost its inhibitory activity while the C40G mutant did not. This suggests that the interchain disulphide bond (Cys133‐Cys141) linking two β‐strands of the six‐strand β‐barrel is essential for ACTI inhibitory activity, while the intrachain disulphide bond (Cys40‐Cys86) connecting the two loops is non‐essential.
Synaptotagmin I is essential for Ca2+‐independent release of neurotransmitter induced by α‐latrotoxin
Authors: Shoji-Kasai Yoko , Yoshida Akira , Ogura Akihiko , Kuwahara Reiko , Grasso Alfonso , Takahashi Masami
Published: July 2002

α‐Latrotoxin causes massive release of norepinephrine from clonal rat pheochromocytoma PC12 cells, in the absence of external Ca2+, by an unknown mechanism. The effect almost disappeared in PC12 variant cells deficient in synaptotagmin I, a synaptic vesicle protein, and was rescued by transfecting the synaptotagmin I gene. These results indicate that synaptotagmin I is essential for the Ca2+‐independent action of α‐latrotoxin in PC12 cells.
Transfection of cells with transforming growth factor‐α leads to cellular resistance to the antiproliferative effects of tumor necrosis factor
Authors: Aggarwal Bharat B. , Pocsik Eva , Ali-Osman Francis , Totpal Klara
Published: July 2002

Tumor necrosis factor (TNF) is a growth‐modulatory cytokine that inhibits the growth of certain cell lines, stimulates the growth of some, and has no effect on the growth of still others. The molecular basis for this differential regulation of growth by TNF is not understood. We postulate that the growth of normal or tumor cells is determined by the balance between growth‐stimulatory and ‐inhibitory signals. In the present study, we demonstrate that the transfection of cells with the transforming growth factor (TGF)‐α gene induces resistance to TNF. Colon carcinoma cell lines that express elevated levels of TGF‐α were also found to be resistant to this cytokine. Exogenous addition of the growth factor was also effective in decreasing the antiproliferative effects of TNF. Transfection of cells with the TGF‐α gene led to downmodulation of TNF receptors but an increase in intracellular glutathione levels. Thus, these results support our hypothesis that expression of growth factors by certain tumor cells can lead to resistance to antiproliferative agents such as TNF.
Antithrombin histidine variants 1H NMR resonance assignments and functional properties
Authors: Fan Bingqi , Turko Illarion V. , Gettins Peter G.W.
Published: July 2002

Three variants of the 57.5 kDa human plasma proteinase inhibitor antithrombin, H1Q, H65C, and H120C, have been expressed in baby hamster kidney cells to permit assignment of the 1H NMR resonances from the three histidines and evaluation of the role of these histidines in heparin binding. The NMR assignments have enabled more definitive interpretation of previous NMR‐based studies of human antithrombin to be made. Although resonances of all three histidines are perturbed by heparin binding, only histidine 120 plays a significant role in the heparin binding site. The perturbations of resonances from histidines 1 and 65 indicate proximity to the heparin binding site and consequent sensitivity to the presence of heparin.
Irreversible light‐induced formation of P680+ and reduced cytochrome b559 in the D1‐D2‐Cyt b‐559 complex at low temperature
Authors: Kaminskaya O.P. , Shuvalov V.A.
Published: July 2002

Cytochrome b
559 in D1‐D2‐Cyt b‐559 complexes from spinach can be photoreduced in the presence of DBMIB at a temperature of 18O–240 K upon continuous illumination. The reduction of Cyt b‐559 is accompanied by oxidation of P680. At 240 K recombination of P680+ and reduced Cyt b‐559 is complete in several seconds. At 220 K and below, the state P680+Cyt b‐559red can be trapped for a long time. This indicates that the photoreduced heme is incapable of electron transfer to P680+ at 220 K and below. On the other hand, the chemically reduced heme of Cyt b‐559 is oxidized by P680+ at 77 K. These results are consistent with the presence of two kinds of Cyt b‐559 hemes in D1‐D2‐Cyt b‐559 complexes which participate in different ways in the photochemical reactions.
Cdc25 is not the signal receiver for glucose induced cAMP response in S. cerevisiae
Authors: Goldberg Doron , Segal Marisa , Levitzki Alexander
Published: July 2002

The Ras/cAMP pathway in the yeast S. cerevisiae couples the cell cycle of this unicellular organism to the availability of nutrients. Glucose derepressed S. cerevisiae cells respond to glucose addition by an intracellular rise in cAMP. In the prevailing model, yeast Ras plays a similar role to that of heterotrimeric G‐proteins coupled to cell surface receptors. A crucial element of this model is that the exchanger, Cdc25 is activated by glucose. Such activation would result in a glucose‐dependent rise in GTP‐bound Ras concentration. We here show, in contrast to this view, that Cdc25 cannot be the receiver of the glucose signal. We suggest that the Ras‐GTP/cyclase complex is the molecular element directly receiving the signal while Cdc25‐dependent exchange constitutes a prerequisite for complex formation.
Presence and significance of minor antenna components in the energy transfer sequence of the green photosynthetic bacterium Chloroflexus aurantiacus
Authors: Mimuro Mamoru , Nozawa Tsunenori , Tamai Naoto , Nishimura Yoshinobu , Yamazaki Iwao
Published: July 2002

Antenna components in the energy transfer processes of a green photosynthetic bacterium Chloroflexus aurantiacus were spectrally investigated by time‐resolved fluorescence spectroscopy at −196°C on intact cells. Besides major antenna components so far reported, three minor components were resolved; those were Bchl c located at 785 nm, the baseplate Bchl a at 819 nm and Bchl a in the B808‐866 complex at 910 nm. The last component was assigned to a longer wavelength antenna closely associated with a reaction center. An additional Bchl c fluorescence component was kinetically suggested to be present, which can be an energy donor to a major Bchl c. Presence of these minor components was signified in terms of (1) increase in the spectral overlap integral and (2) adjustment of the direction of dipole moments in the energy transfer sequence of intact cells.
Inhibition of Moloney murine leukemia virus replication by tyrphostins, tyrosine kinase inhibitors
Authors: Esther Aflalo , Iftach Seri , Esther Priel
Published: July 2002

We have previously shown that topoisomerase I (topo I) antagonist inhibited retrovirus replication. Since tyrphostins, synthetic compounds and protein tyrosine kinases (PTKs) blockers, inhibited topo I activity (manuscript in preparation) we examined their ability to inhibit Moloney murine leukemia virus (Mo‐MuLV) replication. We found that non‐cytotoxic doses of tyrphostin derivatives (AG‐555, AG‐18) blocked or substantially reduced Mo‐MuLV replication in acute or chronically infected NIH/3T3 cells. Our experiments suggest that the antiviral effect of these tyrphostin derivatives was not the result of antiproliferative activity. However, the tyrphostin derivatives used in our present investigation differ in their ability to inhibit Mo‐MuLV replication. Furthermore, as expected from stereospecific competitive inhibitors, the antiviral effect is not a general characteristic of all tyrphostin derivatives, since AG‐213 does not affect Mo‐MuLV replication. Our results indicate that these tyrphostin derivatives may represent a novel class of antiretroviral drugs.
Pertussis toxin‐catalyzed ADP‐ribosylation of GTP‐binding proteins with digoxigenin‐conjugated NAD
Authors: Takei Yoshinori , Takahashi Katsunobu , Kanaho Yasunori , Katada Toshiaki
Published: July 2002

ADP‐ribose moiety containing digoxigenin was transferred by pertussis toxin (IAP) to the α subunit of Gi (Giα) from digoxigenin‐conjugated NAD (DIG‐NAD) in a βγ subunit‐dependent manner. ADP‐ribosylation of Giα with DIG‐NAD plus IAP was inhibited by native NAD. These results indicate that nonradiolabeled DIG‐NAD also serves as the substrate for IAP‐catalyzed ADP‐ribosylation of G proteins. Using DIG‐NAD and fluorescein isothiocyanate‐labeled anti‐digoxigenin antibody, IAP‐sensitive G protein(s) was found to be exist in nuclei as well as plasma membranes of rat liver and HeLa cells. Thus, DIG‐NAD is useful to identify pertussis toxin‐substrate G proteins.
Calcium influx blocks the skeletal muscle acetylcholine receptor α‐subunit gene in vivo
Authors: Huang Chang-Fen , Schmidt Jakob
Published: July 2002

The transcriptional activity of the acetylcholine receptor α‐subunit gene was measured in denervated chick skeletal muscle in response to calcium‐active drugs, using a ribonuclease protection version of the conventional run‐off assay. The L‐channel agonist (−)Bay‐K6844 and the calcium ionophore A23187 mimicked, and the intracellular chelator BAPTA and the calcium channel blockers D600 and nifedipine blocked, the effect of electrostimulation. These results suggest that influx of extracellular calcium is an integral component of the membrane depolarization‐receptor gene inactivation cascade.
Prolactin treatment increases GLUT2 but not the G protein subunit content in cell membranes from cultured neonatal rat islets
Authors: de Mazancourt Philippe , Carneiro Everardo M. , Atwater Illani , Boschero A.Carlos
Published: July 2002

Neonatal rat islets exhibit a reduced secretory response to glucose, compared to adult rat islets. The maturation of the secretory response is stimulated by prolactin (PRL). We show here by immunoblot analysis that PRL increases the β‐cell/liver glucose transporter GLUT2 in membrane fractions from cultured neonatal rat islets. This increase (+86%) may explain, at least in part, the development of a mature glucose response. G proteins modulate insulin secretion from pancreatic β‐cells. We show here by immunoblot analysis that, in contrast to the effect on GLUT2, PRL treatment does not modify the G protein subunits αi2, αi3, αo, αs, αq and β35 and β36, in cultured neonatal islets.