bluuebunny/crossref_metadata_2025 huggingface dataset:
Two rabbit uricase mRNAs and their tissue‐specific expression
Authors: Motojima Kiyoto , Goto Sataro
Published: July 2002

Presence of two uricase mRNA species in rabbit was revealed by cDNA cloning. They were different only in the 5″‐non‐coding region. Their hepatic and extra‐hepatic expression in a tissue‐specific manner was demonstrated by Northern blot and primer extension analyses.
Synergism of type I and type II Interferons in stimulating the activity of the same DNA enhancer
Authors: Vaiman Daniel , Pietrokovsky Samuel , Cohen Batya , Benech Philippe , Chebath Judith
Published: July 2002

Type I and type II interferons (IFNs) can act synergistically to activate the transcription of the 2‐5A synthetase gene. We used in vivo functional assays of sequences from the gene promoter region to determine which DNA segment mediates the gene induction by IFNγ and the synergistic effect. We found that the type I IFN‐inducible enhancer (or IRS) of the 2‐5A synthetase gene also confers inducibility by type II IFN to a reporter CAT gene, though the time course and dose response of the induction by the two IFNs are quite different. A clear synergism of the two IFNs in stimulating the IRS is observed at low doses of the two IFNs.
Carboxylterminal deletion mutants of ribulosebisphosphate carboxylase from Rhodospirillum rubrum
Authors: Morell Matthew K. , Kane Heather J. , Andrews T.John
Published: July 2002

The carboxylterminal octapeptide of ribulosebisphosphate carboxylase from Rhodospirillum rubrum, which lacks small subunits, shows homology to a highly conserved region near the amino terminus of the small subunits of hexadecameric ribulosebisphosphate carboxylases, which are composed of large and small subunits. Truncations of the R. rubrum enzyme, which partially or completely deleted the region of homology, demonstrated that the region is not an important determinant of the catalytic efficiency of the enzyme. A further truncation, which replaced the carboxylterminal 19 amino acid residues with a single terminal leucyl residue, yielded a Rubisco whose substrate‐saturated catalytic rate resembled that of the wild‐type enzyme but which had weaker affinities for ribulose‐P2 and CO2.
Characteristics of a cell‐free assay for the delivery of proteins to the plasma membrane
Authors: Edwardson J.Michael , Daniels-Holgate Phillipa U.
Published: July 2002

We have previously described the reconstitution, in a cell‐free system, of the constitutive delivery of a newly synthesized protein, influenza neuraminidase, to the plasma membrane in BHK cells. Here we report some of the characteristics of this in vitro membrane fusion event. We show that fusion requires ATP hydrolysis, and exploit this requirement to distinguish the time‐course of fusion from that of neuraminidase action. In addition, we present evidence for the occurrence of multiple fusions between hybrid membrane vesicles.
Neural cell adhesion molecule (N‐CAM) in fetal and mature human heart
Authors: Al-Mahdawi Sahar , Shallal Asaad , Wyse Richard K.H.
Published: July 2002

Using Northern blot analysis and immunoblotting techniques we report for the first time, that the neural cell adhesion molecule, N‐CAM, is expressed in human heart. We found several different N‐CAM transcripts in human fetal (13–17 weeks gestation) and mature heart (left ventricle from a 5‐year‐old child). Northern blotting showed that a 5.2 kb transcript was the most abundant and progressively increased with age, both in fetal and mature heart. These transcripts may correspond with the different protein isoforms shown by immunoblotting. We also confirmed the presence of N‐CAM in fetal and mature myocytes by immunohistochemical techniques, using a monoclonal antibody to human N‐CAM. Results demonstrated that N‐CAM is mainly confined to the myocyte cell surface.
A NMR study of mobility in the histone octamer
Authors: Schroth Gary P. , Yau Peter , Imai Brian S. , Gatewood Joe M. , Bradbury E.Morton
Published: July 2002

The histone octamer from chicken erythrocytes was studied in 2 M NaCI using 500 mHz 1H NMR spectroscopy. We compared the spectrum of control octamers with that of octamers isolated from trypsinized nucleosome core particles. We observe that the sharp resonances found in the spectrum of the native octamer disappear completely after trypsinization. Therefore, within the time frame of the NMR experiment, all of the mobile amino acid residues in the histone octamer are found in the well defined trypsin sensitive domains. These results indicate that there is a very clear structural demarcation between the random coil N‐ and C‐terminal tails and the globular domains of the histones.
Activity and gene expression of transglutaminase in guinea pig liver during the postnatal growing phase
Authors: Ikura Koji , Suto Naoki , Sasaki Ryuzo
Published: July 2002

During the postnatal growing phase from birth to 7 weeks old, the cytosolic transglutaminase activity of guinea pig liver increased 3.8‐fold. The enzyme activity in the particulate fraction increased slightly. Immunoblot analyses showed that the postnatal increase in the activity was correlated with an increase in the enzyme protein. The quantity of mRNA of the liver transglutaminase did not change significantly during the postnatal growing phase examined. These results indicated that transglutaminase may be involved in the postnatal development of guinea pig liver and that the amount of transglutaminase in the postnatal liver may be controlled post‐transcriptionally.
Amino acid sequence of pheromone‐inducible surface protein in Enterococcus faecalis, that is encoded on the conjugative plasmid pPD1
Authors: Nakayama Jiro , Nagasawa Hiromich , Isogai Akira , Clewell Don B. , Suzuki Akinori
Published: July 2002

The major pheromone‐inducible protein, PD78, believed to contribute to bacterial conjugation, was purified from Enterococcus (formerly Streptococcus) faecalis cells containing the plasmid pPD1. A cloned EcoRI‐Bg1II 3.6‐kbp fragment of the plasmid pAM351(pPd1::Tn916) contained an open reading frame corresponding to 467 amino acid residues representing PD78. In a central region of the deduced protein, there is a repeated sequence of X‐X‐Pro that is repeated 15 times. This is analogous to the Gin‐Gin‐Pro repeat in the C‐tenninal region of TraD product encoded on the R 100 plasmid in Escherichia coli.
DNA demethylation in erythroleukaemia cells
Authors: Adams R.L.P. , Hanley A. , Rinaldi A.
Published: July 2002

Despite a fall in the proportion of CGs methylated, evidence has not been obtained for significant demethylation of prelabelled DNA when mouse erythroleukaemia cells are induced to differentiate. There is, however, a delay in the methylation of the DNA that is synthesised in the early period of induction, leading to its undermethylation by 30–50% and this may be a contributory cause of the observed fall in CG methylation.
Inhibition of γ‐glutamyl transpeptidase decreases amino acid uptake in human keratinocytes in culture
Authors: Viña Juan R. , Blay Pilar , Ramirez Ana , Castells Antonio , Viña José
Published: July 2002

Acivicin inhibits γ‐glutamyl transpeptidase activity in human keratinocytes in culture. Treatment of these cells with acivicin produces a decrease in the uptake of L‐[U‐14C]alanine, 2‐amino‐[1‐14C]‐isobutyrate, L[U‐14C]leucine and l‐aminocyclopentane‐l‐[14C]carboxylate. D‐[U‐14C]glucose uptake is not affected by the presence of acivicin. These results support, for the first time in vitro, the hypothesis that the γ‐glutarml cycle may be involved in amino acid uptake by human cells.
Paf‐acether‐induced superoxide anion generation in human B cell line
Authors: Leca Gerald , Joly Francine , Vazquez Aimé , Galanaud Pierre , Ninio Ewa
Published: July 2002

Paf‐acether (paf) and lyso phospholipids induced an oxydative burst on EBV‐transfonned B lymphocyte cell line. Superoxide anion formation measured by lucigenin‐dependent chemiluminescence was dependent on both paf concentration and time‐course of challenge. Paf C18:0 at 10 μM was more potent than its C16:0 analogue at the same concentration. Choline‐containing phospholipids with 2‐acyl (long chain) were inactive. The paf antagonists BN 52021 and WEB 2086 structurally unrelated to paf were inactive whereas paf structural analogue CV 3988 inhibited Superoxide formation induced by paf and lysophospholipids. Such a phospholipid‐induced oxydative burst in B cells might exert an effect in the numerous pathophysiological situations where large amounts of paf are produced by phagocytic cells.
Macrophage proteases can modify low density lipoproteins to increase their uptake by macrophages
Authors: Leake David S. , Rankin Sara M. , Collard Jane
Published: July 2002

When low density lipoprotein (LDL) was incubated with sonicated macrophages at acidic pH, its protein moiety was partially degraded by cathepsins B and D. The reisolated LDL was taken up by intact macrophages up to about 20 times as fast as control LDL. LDL proteolysis and its enhanced uptake could be inhibited almost entirely by the selective protease inhibitors leupeptin and pepstatin. If macrophages in atherosclerotic lesions were to release acidic proteases (either by exocytosis or following cell death) and these were to modify LDL, this may help to explain why so much cholesteryl ester accumulates in these cells.
Uni‐site catalysis in thylakoids
Authors: Fromme Petra , Gräber Peter
Published: July 2002

ATP‐hydrolysis was measured with thylakoid membranes during continuous illumination. The concentrations of free and enzyme‐bound ATP, ADP and Pi were measured using either cold ATP, [γ‐32P]ATP or [14C]ATP. The concentration of free ATP was constant, free ADP and enzyme‐bound ATP were below the detection limit. Nevertheless, [γ‐32P]ATP was bound, hydrolyzed and 32Pi was released. The ADP was not released from the enzyme but cold Pi was bound from the medium, cold ATP was resynthesized and released. A quantitative analysis gave the following rate constants: ATP‐binding kATP
= 2·105 M−1s−1, ADP‐release: kADP
<10−2s−1, Pi‐release: kPi
= 0.1 s−1. These rate constants are considerably smaller than under deenergized conditions. The rate constant for the release of ATP can be estimated to be at least 0.2 s−1 under energized conditions. Obviously, energization of the membrane, i.e. protonation of the enzyme leads mainly to a decrease of the rate of ATP‐binding, to an increase of the rate of ATP release and to a decrease of the rate of ADP‐release.
Abnormal behavior of protein kinase C in the human myeloma cell line, RPMI 8226
Authors: Parant Marc R. , Klein Bernard , Vial Henri
Published: July 2002

Protein kinase C activity of the human myeloma cell line, RPMI 8226, was studied after prepurification on DEAE‐cellulose. The total protein kinase activity, eluted at 0.12 M NaCl, was 493 cells, but 38% was associated with membranes. The lipid dependence of cytosolic and membrane activities was only 52% and 21%, respectively. This activity increased with time, to as much as 200% for the membrane fraction after 7 days, whereas lipid dependence and the PDBu binding properties were lost. This modified activity was not due to the extinction of a copurifying endogenous inhibitor nor to classical PKC proteolysis. TPA‐treatment of these eels is accompanied by a rapid, selective and complete loss of lipid‐dependent activity of the cytosol, thus benefiting co‐migrating lipid independent activity, with no membrane fraction recovery or PKM formation.
Action of spermidine, N1‐acetylspermidine, and N8acetylspermidine at apurinic sites in DNA
Authors: Haukanes B.I. , Szajko K. , Helland D.E.
Published: July 2002

The cleavage efficiency of spermidine and its acetyl derivatives (N1‐acetylspermidine and N8‐acetylspennidine) at apurinic sites in DNA were examined by PAGE‐urea analysis. The three polyamines induced different rates of cleavage when compared at 1 mM concentrations. The order of effectiveness were: spermidine <
N
8‐acetylspermidine <
N
1‐acetylspermidine. Thus a decrease in efficiency was observed when the first order aminogroups of spermidine were blocked. The N‐8amino‐group of spermidine was less effective in inducing cleavage at AP‐sites than the N1‐amino‐group. Among several proposed models ofpolyamine‐DNA interactions, our results can best be explained by the model postulated by Liquori et al. [6].
Rat lactate dehydrogenase A and B subunit concentrations are not regulated by mRNA abundance in liver and heart
Authors: Skidmore Annette F. , Beebee Trevor J.C.
Published: July 2002

RNA was isolated from rat liver and heart tissues at various times up to 12 weeks after birth, and probed on slot blots with lactate dehydrogenase A and B cDNA probes. Although the relative abundances of LDH A in liver and LDH B in heart increased substantially in the 12 weeks after birth, mRNAs for both isoenzymes remained remarkably stable in both tissues over the same period. The implications of these observations for the regulation of constitutive gene expression are discussed.
Biosynthesis of acridone alkaloids formation of rutacridone by cell‐free extracts of Ruta graveolens cell suspension cultures
Authors: Maier Walter , Schumann Brigitte , Gröger Detlef
Published: July 2002

Microsomes prepared either by ultracentrifugation or MgCl2 precipitation from cultured Ruta graveolens cells catalyze the condensation of 1,3‐dihydroxy‐N‐methylacridone and isopentenylpyrophosphate or dimethylallylpyrophosphate. In the presence of NADPH and oxygen rutacridone was identified as reaction product. By omission of NADPH glycocitrine‐II is accumulated. The results suggest that at first a prenylated acridone is formed which in turn is cyclized giving the dihydrofuran part of rutacridone.
Low‐density lipoproteins interact with liposome‐binding sites on the cell surface
Authors: Galkina S.I. , Ivanov V.V. , Preobrazhensky S.N. , Margolis L.B. , Bergelson L.D.
Published: July 2002

Under physiological conditions significant amounts of low‐density lipoprotein LDL particles ar taken up by cells independently of specific high affinity LDL receptors (apo‐B receptors). Previously it was established that some cells contain surface sites capable of binding liposomes. We proposed that liposome‐binding sites could contribute to LDL interaction with the cell surface via phospholipid molecules of LDL particles. To check this hypothesis we studied the competitive interaction of human LDL and DPPC liposomes with mouse embryo fibroblasts depleted of apo‐B receptors by preliminary incubation with LDL. We have found that after removal of the liposome‐binding sites from cell lamellae these areas of the cell surface lose their ability to bind LDL.
High‐level expression of enzymatically active bovine leukemia virus proteinase in E. coli
Authors: Andreánsky Martin , Hrušková-Heidingsfeldová Olga , Sedláčeka Juraj , Konvalinka Jan , Bláha Ivo , Ječmen Petr , Hořejši Magda , Štrop Petr , Fábry Milan
Published: July 2002

An E. coli plasmid expressing efficiently an artificial precursor of bovine leukemia virus (BLV) proteinase under transcriptional control of the phage T7 promoter was constructed. The expression product accumulates in the induced E. coli cells in the form of insoluble cytoplasmic inclusions. Solubilization of the inclusions and a refolding step yield almost pure and completely self‐processed proteinase. Purification to homogeneity was achieved by ion‐exchange chromatography and reverse‐phase HPLC. On a preparative scale, a high yield of enzymatically active proteinase was obtained. An initial study using a series of synthetic peptide substrates shows a distinct substrate specificity of BLV proteinase.
O‐H stretching vibration in Fourier transform difference infrared spectra of bacteriorhodopsin
Authors: Chang Chia-Wun , Sekiya Noriko , Yoshihara Kazuo
Published: July 2002

FTIR difference spectroscopic studies of M intermediate and LA bacteriorhodopsin in the O‐H stretching region show bands at 3671 and 3641 cm−1, respectively. The O‐H stretching bands in this region may reflect protonation‐deprotonation changes or environmental change in the tyrosine residues in bR.
In vivo promoter activity of the synthetic Pribnow box
Authors: Koroleva O.N. , Drutsa V.L.
Published: July 2002

The synthetic polydeoxyribonucleotides TGCA(TTATAATGCA), ??? a containing the consensus Pribnow box, were inserted upstream of the promoterless gal operon in a specially constructed plasmid. Using the MacConkey agar indicator technique it has been show that these inserts are able to induced an efficient in vivo expression of the gal operon.
Influence of thyroid hormone on ADP‐ribosylation of nuclear proteins in cultured GH1 cells
Authors: Aranda Ana , Copp Richard P. , Pascual Angel , Samuels Herbert H.
Published: July 2002

We present evidence that T3 can alter the ADP‐ribosylation of chromatin associated proteins. Nuclei from GH1 cells were incubated with [adenylate‐32P]NAD and the radioactivity incorporated into histone and non‐histone proteins was quantitated and analyzed by gel electrophoresis and autoradiography. Incubation of GH1 cells for 24 h with T3 lowered by 40–70% the [32P]ADP‐ribose incorporated into nuclear proteins. However, incubation for 3 h with T3 resulted in a simulation instead of a decrease of in vitro [32P]ADP‐ribose incorporation. The major ADP‐ribosylated component electrophoresed as a 120 000 molecular mass non‐histone protein, and radiolabeled histones were also observed. The same protein species were observed for all the experimental groups and T3 affected the extent of ADP‐ribosylation but did not alter the sedimentation of the [32P]ADP‐ribosylated components excised from chromatin after micrococcal nuclease digestion.
Actinomycin D decreases protein kinase C content and induces neuritogenesis in neuroblastoma cells
Authors: Miñana Maria-Dolores , Felipo Vicente , Grisolía Santiago
Published: July 2002

We have previously reported that inhibition of protein kinase C induces differentiation of neuroblastoma cells in culture. It is shown now that actinomycin D, a well known inhibitor of DNA synthesis, reduces selectively the content of protein kinase C and induces neuritogenesis in Neuro 2a cells in culture.
Electron microscopic and biochemical evidence that proline‐β‐naphthylamidase is composed of three identical subunits
Authors: Takahashi Takayuki , Nishigai Masaaki , Ikai Atsushi , Takahashi Kenji
Published: July 2002

Electron microscopy of pig intestinal proline‐β‐naphthyltamidase revealed that the enzyme is composed of 3 subunits, which are assembled in a trifoliolate shape, At pH 4.5 and 4°C, the enzyme dissociates reversibly into active subunits in 4h. Dissociation also occurs at higher pHs when the enyzme concentration is very low. The activity per mg protein of the native, trimeric enzyme is about 2.5‐fold higher than that of the dissociated enzyme.
On the functional form of transcortin‐recognizing subunit of transcortin membrane receptor
Authors: Krupenko Sergey A. , Avvakumov George V. , Strel'chyonok Oleg A.
Published: July 2002

Complex formation between transcortin and the 20 kDa sialoglycoprotein from the plasma membrane of human decidual endometrium (presumably a transcortin‐recognizing subunit of transcortin membranes receptor) was studied using cross‐linking reagents. The action of 1,5‐difluoro‐3,4‐dinitrobenzene (DFDNB) on a solution of 125I‐labelled 20 kDa sialoglycoprotein and unlabelled transcortin resulted in the formation of two 125I‐containing species that corresponded to covalently linked complexes of one transcortin molecule and either 2 or 4 molecules of the labelled membrane sialoglycoprotein. Only the latter complex was observed when the endometrium membranes were incubated with [25I]transcortin and treated with DFDNB. This suggests that the functional form of transcortin‐recognizing subunit of the membrane receptor is a tetramer.
Role of glycine‐82 as a pivot point during the transition from the inactive to the active form of the yeast Ras2 protein
Authors: Kavounis Constantin , Verrotti Arturo C. , De Vendittis Emmanuele , Bozopoulos Anastasios , Di Blasi Francesco , Zahn Regina , Crechet Jean Bernard , Parmeggiani Andrea , Tsernoglou Demetrios , Fasano Ottavio
Published: July 2002

Ras proteins bind either GDP or GTP with high affinity. However, only the GTP‐bound form of the yeast Ras2 protein is able to stimulate adenylyl cyclase. To identify amino acid residues that play a role in the conversion from the GDP‐bound to the GTP‐bound state of Ras proteins, we have searched for single amino acid substitutions that selectively affected the binding of one of the two nucleotides. We have found that the replacement of glycine‐82 of the Ras2 protein by serine resulted in an increased rate of dissociation of Gpp(NH)p, a nonhydrolysable analog of GTP, while the GDP dissociation rate was not significantly modified. Glycine‐82 resides in a region that is highly conserved between the yeast and human proteins. However, this residue is structurally distant from residues that participate in the binding of the nucleotide, as determined from the crystal structure of the human H‐ras gene product. Therefore, the ability of the nucleotide binding site to discriminate between GDP and GTP is dependent not only on residues that are spatially close to the nucleotide, but also on distant amino acids. This is in agreement with the role of glycine‐82 as a pivot point during the transition from the GDP‐ to the GTP‐bound form of the Ras proteins.
Comparison of the amino acid and nucleotide sequences between human and two guinea pig major basic proteins
Authors: Aoki Ikuo , Shindoh Yutaka , Nishida Tsutomu , Nakai Satoru , Hong Yeong-Man , Mio Mitsunobu , Saito Tadayuki , Tasaka Kenji
Published: July 2002

By means of reverse‐phase HPLC, 2 different proteins were obtained from apparently purified pig cosinophil major basic protein (MBP) and these proteins were named GMPB1 and GMBP2. It was revealed that these 2 components of MBP have similar molecular weights and pI values although the amino acid compositions were slightly different. In the previous study, we cloned and sequenced GMPB1 cDNA. Here we obtained another clone by plaque hybridization using a screening probe synthesized by means of polymerase chain reaction. After sequencing, it became apparent that this clone corresponded to GMBP2. As in the case of GMBP1, the cDNA of GMBP2 encoded pre‐proGMBP2 with 3 domains; signal peptide, acidic pro‐portion, and mature GMBP2. By comparing the sequences of GMBP1 and GMBP2, it was revealed that the proteins were quite similar to each other. In addition, their sequences also resembled those of human MBP, especially in the basic domain of mature protein: but no such similarity existed in the pro‐portion. Although the molecular weights determined by SDS‐PAGE of guinea pig and human MBPs were 11 000 and 9 300, respectively, the calculated molecular weights of these 3 MBPs were all 13.8 kDa. The calculated pI values of GMBP1, GMBP2 and human MBP were 11.7, 11.3 and 11.6, respectively. By means of Harr plot analysis, it was revealed that the amino acid sequences, not only in signal peptides but also in the bisic domains of mature proteins, were well conserved between guinea pig and human MBPs.
Role of a GC‐rich motif in transcription regulation of the adenovirus type 2 IVa2 promoter which lacks typical TATA‐box element
Authors: Tamura Taka-aki , Mikoshiba Katsuhiko
Published: July 2002

Promoter of the adenovirus type 2 IVa2 (IVa2) gene does not have a TATA‐box element, hence it is considered to be a model of cellular TATA‐less promoters. In vitro transcription of the IVa2 promoter in HeLa cell extracts identified a proximal cis‐element around −40 relative to the transcription start site. DNase I footprint analysis revealed the presence of a DNA‐binding factor which interacts with sequences ACCCCTCCCACTTAG at −50. Competition footprint analysis and sequence comparisons showed that the IVa2 proximal promoter element is equivalent to the GC‐box, the Spl‐binding site.
Structural analysis by circular dichroism of some enzymes involved in plant cell wall degradation
Authors: Goodenough P.W. , Clark D.C. , Durrant A.J. , Gilbert H.J. , Hazlewood G.P. , Waksman G.
Published: July 2002

Three enzymes which degrade different polysaccharide components of plant cell walls have been characterized by circular dichroism (CD). A bacterial endoglucanase, which in the native state forms part of a multiprotein cellulase complex, showed a tendency to form aggregates as measured by CD. Depending on its degree of aggregation, this enzyme displayed between 50% and 100% helical structure, whereas a bacterial xylanase and a fungal polygalacturonase exhibited more β‐sheet structure. The polygalacturonase was apparently devoid of helical structure.
Intrinsic tryptophan phosphorescence as a marker of conformation and oxygen diffusion in purified cytochrome oxidase
Authors: Papp Sandor , King Tsoo E. , Vanderkooi Jane M.
Published: July 2002

Cytochrome oxidase exhibits phosphorescence from trytophan in aqueous solution in the absence of oxygen. The lifetime for the resting reduced enzyme suspended in Tween‐20 is around 30 ms at pH 8. The lifetime is longest between pH 7 and 8 and decreases with lowering of pH. Oxygen quenches the phosphorescence with a Stern‐Volmer quenching constant of ∼5 × 107 M−1 s−1 at 5°C whereas cytochrome c has no effect. We interpret these results to indicate that room temperature tryptophan phosphorescence arises from trytophan(s) in structured region(s) remote from the hemes and that the protein does not impose a significant barrier for the diffusion of oxygen.
The localization of the cAMP‐dependent protein kinase phosphorylation site in the platelet rat protein, rap 1B
Authors: Fischer Thomas H. , Collins John H. , Gatling Margaret N. , White Gilbert C.
Published: July 2002

Rap1B is a low molecular weight G protein which is phosphorylated by cAMP‐dependent protein kinase. In order to identify the site of phosphorylation by cAMP‐dependent protein kinase, purified rap1B from human platelets was phosphorylated and subjected to limited proteolysis with trypsin. Single digestion fragment containing the phosphorylation site was obtained and purified by reversed‐phase HPLC. Sequence analysis of the phosphorylated digestion fragment demonstrated that the sequence of the phosphorylation site was ‐Lys‐Lys‐Ser‐Ser‐. This sequence is near the carboxyl terminus and is adjacent to the site of membrane attachment of the protein.
Evidence for a role of phosphofructokinase and tRNA in the polyribosome disaggregation of amino acid deficiency
Authors: Rabinovitz Marco
Published: July 2002

The activity of rabbit muscle phosphofructokinase was inhibited by transfer ribonucleic acid. This inhibition was reduced by inclusion of an amino‐acyl‐tRNA charging system. The results are discussed in terms of the loss of ATP in amino acid deprived cells and in the critical role of fructose 1,6‐diphosphate in peptide chain initiation.
Change in charge of an unvaried heme contact residue does not cause a major change of conformation in cytochrome c
Authors: Thurgood Andrew G.P. , Pielak Gary J. , Cutler Robert L. , Davies Anne M. , Greenwood Colin , Grant Mauk A. , Smith Michael , Williamson David J. , Moore G.R.
Published: July 2002

The only significant area of conformational change detected was at residues 39 and 58, close to the site of modification. Therefore the redox potential change accompanying the modification [1988, Biochemistry 28, 3198–3197] appears to be a direct consequence of the altered side‐chain of residue 38 and not a result of secondary conformational changes induced by the modification.
Heterogeneity in the Xenopus ribosomal transcription factor xUBF has a molecular basis distinct from that in mammals
Authors: Bachvarov Dimcho , Normandeau Marc , Moss Tom
Published: July 2002

The Xenopus polymerase I transcription factor xUBF is an HMG‐box protein which has been purified as two polypeptides of ∼82 and 85 kDa. Recently a cDNA sequence predicted an xUBF protein (xUBF1) of 677 amino acids (79 kDa) containing five tandem HMG‐boxes. Here a second and distinct xUBF cDNA has been isolated and characterised. This cDNA codes an xUBF protein (xUBF2) of 701 amino acids (82 kDa), having 93% homology with xUBF1 but containing an insertion of 22 amino acids between HMG‐boxes 3 and 4. In vitro translation of synthetic mRNAs derived from the xUBF1 and 2 cDNAs was used to show that the electrophoretic mobility of the gene products accounted for the major xUBF molecular weight heterogeneity noted in vivo. It is also shown that the Xenopus laevis genome contains 3 or 4 distinct loci which hybridise with xUBF coding sequences, leaving open the possibility of yet further unrecognised heterogeneity in xUBF.
The lac repressor and its N‐terminal headpiece can bind a mini‐operator containing a hairpin loop made of a hexaethylene glycol chain
Authors: Maurizot J.C. , Chevrie K. , Durand M. , Thuong N.T.
Published: July 2002

The binding of the lac repressor and the lac repressor N‐terminal headpiece to a mini‐operator with a hairpin loop made of a hexaethylene glycol chain was investigated using circular dichroism spectroscopy. The Iac repressor's headpiece binds to the modified mini‐operator with the same affinity as to a mini‐operator of the same sequence without the hexaethylene glycol loop. The conformational effect due to the binding is not affected by the presence of the hexaethylene loop. It is also shown that the entire lac repressor binds to this modified mini‐operator inducing a conformational change.
A PAF‐acetylhydrolase activity in Tetrahymena pyriformis cells
Authors: Tselepis Alexandros D. , Lekka Marilena E. , Tsoukatos Demokritos
Published: July 2002

Our study provides evidence for the existence of an acylhydrolase activity in Tetrahymena pyriformis cells, capable of hydrolizing the sn‐2 ester bond of the PAF molecule. This activity is mainly distributed in the microsomal fraction (76.5% of total) and has properties similar to the mammalian PAF‐acetylhydrolase since it is Ca2+‐independent, acid‐labile, is inhibited by DFP and PMSF but it is not affected by egg yolk phosphatidylcholine. This microsomal acylhydrolase has apparent Km
and Vmax
values of 1.56 μM and 373 pmols ‐ mg ‐ min respectively. This is the first report of the existence of a PAF‐acetylhydrolase activity in a non‐mammalian cell.
Characterization of a tissue‐type plasminogen activator from porcine urine
Authors: Zacharias Ute , Will Horst
Published: July 2002

Porcine urine, unlike human urine, does not contain detectable amounts of urokinase‐type plasminogen activator (u‐PA). The plasminogen activator present in porcine urine is of tissue‐type (t‐PA) as identified by the following criteria. (1) Porcine urine PA exhibits an M
r of 65 000 similar to the M
r of human t‐PA (64‐70 000) but distinct from the M
r of human u‐PA (55 000). (2) Antibodies against human t‐PA bind and inhibit crude and purified porcine urine PA, while human u‐PA‐specific antibodies do not react with porcine urine PA. (3) Plasminogen activation by porcine urine PA is markedly stimulated in the presence of fibrinogen fragments. (4) Porcine urine PA activity is not affected by concentration of amiloride substantially suppressing human u‐PA activity.
Cloning and sequence analysis of pituitary cDNA encoding the β‐subunit of Xenopus proteasome
Authors: van Riel Maarten C.H.M. , Martens Gerard J.M.
Published: July 2002

The proteasome is a multicatalytic proteinase composed of a number of non‐identical subunits. A Xenopus pituitary cDNA was isolated and found to code for the β‐subunit of proteasome. The amino acid sequence deduced from the open reading frame consisted of 215 amino acid residues with a calculated molecular weight of 23 979. A comparative structural analysis indicated that the proteasome subunits can be divided into two groups with the same evolutionary origin. One group consists of subunits with an N‐terminally blocked residue and includes components C2, C3, C8 and C9, while the second group of non‐blocked proteins includes components C5 and the β‐subunit.
The 65‐kDa protein from pig heart A new substrate for Clostridium botulinum ADP‐ribosyltransferase (exoenzyme C3)
Authors: Hoffenberg Simon I. , Rybin Vitalyi O. , Efimenko Alexander N. , Kurochkin Iliya N. , Tkachuk Vsevolod A.
Published: July 2002

In the pig heart sarcolemma, a 65 kDa protein is found to be ADP‐ribosylated by Clostridium botulinum ADP‐ribosyltransferase (exoenzyme C3). ADP‐ribosylation of this protein is regulated by guanyl nucleotides and cytosol factor in a fashion similar to that for other C3 substrates. The new exoenzyme C3 substrate was partially purified. This protein is supposed to be a GTP‐binding one.
Structure and expression of subunit A from the bovine chromaffin cell vacuolar ATPase
Authors: Pan Ying-Xian , Xu Jin , Strasser Jane E. , Howell Michael , Dean Gary E.
Published: July 2002

Subunit A of the vacuolar H+‐ATPase class is thought to be responsible for the ATP hydrolysis which drives proton‐pumping. We report here the cloning and sequence determination of the first mammalian cDNA encoding a bovine vacuolar ATPase subunit A from an adrenal medulla cDNA library. Northern blots of bovine adrenal medulla RNA reveal a message of approximately 3.8 kb. The predicted peptide sequence, consisting of 618 amino acids with a calculated molecular weight of 68397 daltons, is similar to the sequences of the three known subunit A proteins. β‐Galactosidase‐subunit A fusion proteins were immuno‐decorated by an antiserum raised to the subunit A protein from corn coleoptile vacuoles.
Between the ground‐ and M‐state of bacteriorhodopsin the retinal transition dipole moment tilts out of the plane of the membrane by only 3°
Authors: Otto Harald , Heyn Maarten P.
Published: July 2002

The orientation of the transition dipole moments in the ground state and the M‐intermediate of bacteriorhodopsin were determined by time‐resolved and steady‐state polarized absorption spectroscopy on samples of oriented immobilized purple membranes. The angle between the transition dipole moment and the membrane normal decreases from 66.8±0.5° in the all‐trans ground state to 64.1±0.8° in the 13‐cis M‐state. The light‐induced isomerization of the chromophore is thus accompanied by an orientational change of only about 3° out of the plane of the membrane. The absorption anisotropy at 410 nm remains constant over more than 4 decades of time covering both the rise and decay of M. Conformational changes accompanying a sequential M1→M2 transition thus do not affect the chromophore orientation.
Phorbol 12‐myristate‐13‐acetate (PMA) stimulates a differential expression of cholecystokinin (CCK) and c‐fos MRNA in a human neuroblastoma cell line
Authors: Monstein Hans-Jürg , Folkesson Ronnie
Published: July 2002

Regulation of cholecystokinin (CCK) and the proto‐oncogene c‐fos mRNA expression was studied in the human neuroblastoma cell line SK‐N‐MC. Cells were treated either with the tumor promoting phorbolester phorbol‐12‐myristate‐13‐acetate (PMA), the phosphodiesterase inhibitor isobutyl‐methylxanthine (IBMX), which results in an elevated intracellular cyclic AMP (cAMP) level, or with a combination of PMA and IBMX. The level of CCK and c‐fos mRNA was determined by Northern‐blot analysis with CCK and c‐fos specific antisense RNA probes after 4–24 h of drug treatment. Treatment with PMA and IBMX for 4–24 hours transiently raised the CCK MRNA level ∼1.5–3.5 times compared to the controls, and the combination PMA and IBMX had an additive effect and elevated CCK MRNA abundance 1.5–6.5 times. Under the same experimental conditions, both PMA and IBMX elevated the c‐fos MRNA level ∼3–5.5 times. The drug combination showed a pronounced synergistic effect and raised the c‐fos mRNA level ∼3–20 times as compared to controls. Apparently, CCK and c‐fos mRNA expression appears to be regulated by similar protein kinase C (PKC) and cAMP‐dependent mechanisms in SK‐N‐MC cells.
Structure‐function analysis of the human integrin VLA‐4 (α4/β1)
Authors: Pulido Rafael , Campanero Miguel R. , García-Pardo Angeles , Sánchez-Madrid Francisco
Published: July 2002

The structure‐function relationship of the human integrin VLA‐4 (α4/β1; CD49d/CD29), has been studied in the human B‐cell line Ramos by immunochemical and functional analysis. Ramos cells expressed the 150‐kDa non‐proteolyzed form of the α4 chain, which could be digested upon mild trypsin treatment to generate the 80‐ and 65‐kDa proteolyzed forms, as well as α4 polypeptides of 55 and 50 kDa. In addition, treatment of Ramos cells with high doses of pronase predominantly yielded the 55‐ and 50‐kDa α4 peptides. The trypsin‐generated 80‐ and 65‐kDa α4 polypeptides, but not the 55‐ and 50‐kDa fragments, were able to associate with the β1 chain. Distinct anti‐VLA‐4 mAb against four different α4 epitopes, referred to as epitopes A, B1, B2, and C, recognized the 150‐kDa α4 chain both associated or non‐associated with the β1 chain. The α4 proteolytic forms of 80, 65 and 50 kDa were precipitated by the anti‐α4 mAb directed against the four different α4 epitopes. On the other hand, the 55‐kDa α4 peptide was present in precipitates from anti‐α4 mAb specific for epitopes A, B1 and C, but absent in precipitates from the anti‐α4 mAb specific for epitope B2. The different adhesive capacities of the VLA‐4 integrin, namely the interaction with a 38‐kDa fibronectin fragment containing the CS‐1 region of plasma fibronectin (Fn‐38), the binding to the vascular cell adhesion molecule‐1 (VCAM‐1), or the ability to mediate the anti‐α4‐induced cell aggregation, were not altered on VLA‐4 from cells upon mild trypsin treatment, when compared to non‐treated cells. However, the 55‐ and 50‐kDa α4 forms generated by high‐dose pronase cell treatment, failed to mediate cell interaction with Fn‐38 or VCAM‐1 ligands, and cell aggregation could not be triggered through VLA‐4 under these conditions.
Biochemical ethanol effects affected by a non‐steroidal anti‐inflammatory drug
Authors: de Piña Marta Zentella , Hernández-Tobías Aída , Saldaña-Balmori Yolanda , Díaz-Belmont Armando , Piña Enrique
Published: July 2002

The non‐steroidal anti‐inflammatory drug, piroxicam, prevents the hepatic increase of triacylglycerols and malondialdehyde resulting from the acute intoxication of rats with ethanol. In addition, in the intoxicated rats, piroxicam consistently produces a decrease in the levels of blood ethanol in comparison with control animals. It is suggested that the anti‐inflammatory compound stimulates ethanol oxidation.
Galanin reduces PDBu‐induced protein phosphorylation in rat ventral hippocampus
Authors: La Porta Caterina , Bianchi Roberto , Sozzani Silvano , Bartfai Tamas , Consolo Silvana
Published: July 2002

The effect of galanin(GAL) on basal and phorbol‐12,13‐dibutyrate (PDBu) induced protein phosphorylation in rat ventral hippocampal miniprisms was investigated. GAL (0.5, 1 and 2μM) inhibited PDBu stimulation in a concentration‐dependent manner without altering basal protein phosphorylation. This inhibitory effect was prevented by the GAL antagonist galantide. GAL did not affect either the activity of protein kinase C (PKC) from rat brain or basal phosphorylation in ventral hippocampal hippogenates, suggesting that it did not directly modulate PKC activity, Depolarization of miniprisms from ventral hippocampl by 18 mM K+ prevented the effect of GAL on PDBu‐induced phosphorylation. The results indicate that GAL indirectly regulates neuronal protein phosphorylation by a GAL receptor‐mediated action.
Arachidonic acid induces phosphorylation of an 18 kDa protein in electrically permeabilised rat islets of Langerhans
Authors: Basudev Harsha , Jones Peter M. , Persaud Shanta J. , Howell Simon L.
Published: July 2002

Arachidonic acid (AA) was shown to induce concentration‐dependent, calcium‐independent, in situ phosphorylation of a protein of approximate molecular weight 18 kDa in electrically permeabilised rat islets of Langerhans. This protein did not appear to be a substrate for protein kinase C (PKC) since stimulation of PKC by 4β phorbol myristate acetate (4β PMA) did not result in 32P incorporation into an 18 kDa protein, and since AA‐induced phosphorylation was observed in islets in which PKC had been down‐regulated by prolonged exposure of islets to 4β PMA. These results suggest that AA stimulates protein phosphorylation by a mechanism other than PKC activation.
The oligosaccharides in a recombinant hepatitis B virus surface antigen (HBsAg) carrying the pre‐S2 region derived from yeast
Authors: Kobayashi Makoto , Ban Junko , Asano Tsuneo , Utsunomiya Midori , Kusumoto Shunji , Nishi Kiyoshi , Kato Koichi
Published: July 2002

The N‐ and O‐linked oligosaccharides in a yeast‐derived HBsAg M protein (pre‐S2 + S) were analyzed. Two major structures of the N‐linked oligosaccharides bound to residue Asn4 were determined to be high‐mannose type oligosaccharides. Man7GlcNAc2 and Man8GlcNAc2, by two‐dimensional sugar mapping of the corresponding pyridylamino oligosaccharides. Peptide mapping of the M protein, sequence analysis of the glycopeptides after β‐elimination under reducing conditions and sugar‐composition analysis revealed that the O‐linked oligosaccharides were composed solely or mannose and bound to residue Ser5, Thr6, Thr7, Ser27, Ser28, Ser29 and Thr31 in the pre‐S2 region.
Nucleotide sequence of a cDNA encoding human tumor necrosis factor β from B lymphoblastoid cell RPMI 1788
Authors: Matsuyama Noriko , Okawa Noriyuki , Tsukii Yuji , Endo Tohru , Kaji Akira
Published: July 2002

Published sequences of cDNA for human tumor necrosis factor β (TNF‐β) have a discrepancy within the coding region as well as exon 1. To resolve these discrepancies we have re‐isolated TNF‐β cDNA from the human B cell lymphoblastoid cell line, RPMI 1788, and determined its DNA sequence. Results indicate that amino acid 26 is threonine (Thr) instead of asparigine (Asn). In contrast to published sequences, the sequence of the exon 1 region corresponded to the genomic sequence of TNF‐β. From our studies we conclude that the TNF‐β gene of the human B cell lymphoblastoid cell line, RPMI 1788, is homologous with respect to the TNF‐β gene.
Proteolysis of Bacillus stearothermophilus IF2 and specific protection by fMet‐tRNA
Authors: Severini Manuela , Choli Theodora , La Teana Anna , Gualerzi Claudio O.
Published: July 2002

Translation initiation factor IF2 from Bacillus steaiothermophilus (741 amino acids, M
r 82,043) was subjected to trypsinolysis alone or in the presence of fMet‐tRNA. The initiator tRNA was found to protect very efficiently the Arg308‐Ala309 bond within the GTP binding site of IF2 and, more weakly, three bonds (Lys146‐Gln147, Lys154‐Glu155 and Arg519‐Ser520). The first two are located at the border between the non‐conserved, dispensable (for translation) N‐terminal portion and the conserved G‐domain of the protein, the third is located at the border between the G‐ and C‐domains. Since IF2 is known to interact with fMet‐tRNA through its protease‐resistant C‐ (carboxyl terminus) domain, the observed protection suggests that, upon binding or fMet‐tRNA, long‐distance tertiary interactions between the IF2 domains may take place.
Relationship of neural cell adhesion molecules (N‐CAMs) with adenylate cyclase
Authors: Lipkin V.M. , Surina E.A. , Petukhova G.V. , Petrov V.M. , Mirzoeva S.F. , Rakitina T.V.
Published: July 2002

Bovine brain cDNA cloned earlier and attributed to calmodulin‐independent adenylate cyclase encodes the neural cell adhesion molecule (N‐CAM). The expression of N‐CAM mRNA in Xenopus laevis oocytes increases their basal adenylate cyclase activity. Polyclonal antibodies against synthetic peptide A, VAENQQGKSKAAHFV (664–678 amino acid residues of bovine N‐CAM), containing sequence AXXXXGKS which is homologous to the nucleotide‐binding consensus sequence GXXXXGKS, inhibit the adenylate cyclase activity. A close relationship appears to exist between adenylate cyclase and N‐CAM.
Inositol trisphosphate‐induced hyperpolarization in rat dorsal root ganglion neurons
Authors: Wang Z. , Ypey D.L. , Van Duijn B.
Published: July 2002

Inositol 1,4,5‐trisphosphate (1,4,5‐InsP3) was perfused into rat dorsal root ganglion (DRG) neurons by whole‐cell patch‐clamp electrodes, while measuring the membrane potential. This operation evoked a transient (2–3 min) membrane hyperpolarization of about −15 mV (from −42 mV) followed by a depolarization. The membrane hyperpolarization was abolished when 30 mM EGTA was perfused together with 1,4,5‐InsP3 or when 0.2 mM quinine was added to the bath solution. The hyperpolarizing response was enhanced when a low‐Ca2+ EGTA‐free intracellular solution was used. Two InsP2 isomers induced a different response. Our results suggest that the hyperpolarization is due to 1,4,5‐InsP3‐induced Ca2+ release which may trigger Ca‐sensitive K+ channels to open. Present results show that cultured DRG neurons are able to respond to 1,4,5‐InsP3 perfusion in the whole‐cell configuration.
Natural abundance 15N NMR assignments delineate structural differences between intact and reactive‐site hydrolyzed Cucurbita maxima trypsin inhibitor III
Authors: Krishnamoorthi Ramaswamy , Nemmers Sylvia , Tobias Brian
Published: July 2002

15N NMR assignments were made to the backbone amide nitrogen atoms at natural isotopic abundance of intact and reactive‐site (Arg5—Ile6) hydrolyzed Cucurbita maxima trypsin inhibitor III (CMTI‐III and CMTI‐III*, respectively) by means of 2D proton‐detected heteronuclear single bond chemical shift correlation (HSBC) spectroscopy, utilizing the previously made sequence‐specific 1H NMR assignments (Krishnamoorthi et al. (1992) Biochemistry 31. 898–904). Comparison of the 15N chemical shifts of the two forms of the inhibitor molecule revealed significant changes not only for residues located near the reactive‐site region, but also for those distantly located. Residues Cys3, Arg5, Leu7, Met8, Cys10, Cys16, Glu19, His25, Tyr27, Cys28 and Gly28 showed significant chemical shift changes ranging from 0.3 to 6.1 ppm, thus indicating structural perturbations that were transmitted throughout the molecule. These findings confirm the earlier conclusions based on 1H NMR investigations.
Transcriptional activation of the gene for the large subunit of human m‐calpain by 12‐o‐tetradecanoyl‐phorbol‐13‐acetate
Authors: Hata Akiko , Ohno Shigeo , Suzuki Koichi
Published: July 2002

The effect of the treatment of HeLa cells with a tumor‐promoting phorbol ester, 12‐o‐tetradecanoyl‐phorbol‐13‐acatate (TPA) on the expression of the genes for the calpain family has been examined. Among the mRNAs for the calpain family, only the mRNA for the large subunit of human m‐calpain (calpain mL) was specifically induced by treatment of cells with TPA, suggesting its specific function in response to cellular stimuli. The effect of TPA on the expression of the calpain mL gene was further examined using fusion genes containing the promoter/enhancer region of the calpain mL gene fused upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene, showing that the promoter/enhancer sequence of the calpain mL gene contains a cis‐acting element which responds to TPA and activates transcription of the downstream sequence.
Effects of antiarrhythmic drugs on phospholipid metabolism in Jurkat T cells The potassium channel blocker, clofilium, specifically increases phosphatidylserine synthesis
Authors: Aussel Claude , Pelassy Claudette
Published: July 2002

Five antiarrhythmic drugs (bretylium, clofilium, propranolol, N‐acetylprocainamide and amiodarone) were tested for their ability to modify phospholipid metabolism in Jurkat T lymphocytes. The five drugs, decreased in a dose‐dependent mode the biosynthesis of both phosphatidylcholine and phosphatidylethanolamine, this effect was essentially due to impairment of either choline or ethanolamine uptake by the cells. The efficiency of the drugs to inhibit phosphatidylcholine and phosphatidylethanolamine synthesis was in the order: clofilium > amiodarone ⪢ propranolol = bretylium >
N‐acetylprocainamide. The IC50 varied from 3–5 μM for clofilium to >200 μM for N‐acetylprocainamide. In contrast, only clofilium, a voltage‐gated K+‐channel blocker, was able to increase phosphatidylserine synthesis with an EC50 = 50 μM. The effect of clofilium on phosphatidylserine synthesis thus mimics the effect of three other K+‐channel blockers, quinine, 4‐aminopyridine and tetraethylammonium, suggesting close relationships between phosphatidylserine synthesis and K+ channel activity.