bluuebunny/crossref_metadata_2025 huggingface dataset:
Charybdotoxin and noxiustoxin, two homologous peptide inhibitors of the K+(Ca2+) channel
Authors: Valdivia Hector H. , Smith Jeffrey S. , Martin Brian M. , Coronado Roberto , Possani Lourival D.
Published: July 2002

We show that noxiustoxin (NTX), like charybdotoxin (CTX) described by others, affects Ca2+‐activated K+ channels of skeletal muscle (K+(Ca2+) channels). Chemical characterization of CTX shows that it is similar to NTX. Although the amino‐terminal amino acid of CTX is not readily available, the molecule was partially sequenced after CNBr cleavage. A decapeptide corresponding to the C‐terminal region of NTX shows 60% homology to that of CTX, maintaining the cysteine residues at the same positions. While CTX blocks the K+(Ca2+) channels with a K
d of 1–3 nM, for NTX it is approx. 450 nM. Both peptides can interact simultaneously with the same channel. NTX and CTX promise to be good tools for channel isolation.
Extremely low frequency pulsed electromagnetic fields increase interleukin‐2 (IL‐2) utilization and IL‐2 receptor expression in mitogen‐stimulated human lymphocytes from old subjects
Authors: Cossarizza A. , Monti D. , Bersani F. , Paganelli R. , Montagnani G. , Cadossi R. , Cantini M. , Franceschi C.
Published: July 2002

The effects of the exposure of mitogen‐stimulated human lymphocytes from aged subjects to low‐frequency pulsed electromagnetic fields (PEMFs) were studied by measuring the production of interleukin‐2 (IL‐2) and the expression of IL‐2 receptor. PEMF‐exposed cultures that presented increased [3H]thymidine incorporation showed lower amounts of IL‐2 in their supernatants, but higher percentages of IL‐2 receptor‐positive cells and of T‐activated lymphocytes. Taken together, these data suggest that PEMFs were able to modulate mitogen‐induced lymphocyte proliferation by provoking an increase in utilization of IL‐2, most likely acting on the expression of its receptor on the plasma membrane.
Enzyme inhibition by dipeptides containing 2,3‐methanophenylalanine, a sterically constrained amino acid
Authors: Ogawa Tomohisa , Yoshitomi Haruko , Kodama Hiroaki , Waki Michinori , Stammer Charles H. , Shimohigashi Yasuyuki
Published: July 2002

Both isomers of (E)‐2,3‐methanophenylalanine (▽EPhe), a sterically restricted amino acid, were incorporated into peptides in order to examine their possible enzyme inhibitory activity. Both (2R,3S)‐ and (2S,3R)‐▽EPhe‐Phe(or Leu)‐OMe were found to inhibit effectively the hydrolysis of Ac‐Tyr‐OEt by chymotrypsin in a competitive manner. The ester groups of these dipeptides were quite resistant to chymotrypsin hydrolysis, and the ▽EPhe‐Phe peptide bond was also entirely stable. The inhibition constant (K
i) of the most potent dipeptide of H‐(2R,3S)‐▽EPhe‐Phe‐OMe was 0.16 mM at 25°C. The inhibitory action of ▽Phe‐containing peptides was found to depend on the configuration of the ▽Phe residue. The electrophilic nature of the cyclopropane ring which is conjugated with both the phenyl ring and the ester carbonyl group appears to be relevant to the inhibitory activity. Fully irreversible inactivation of chymotrypsin was achieved by its incubation with H‐(2R,3S)‐▽EPhe‐Leu‐OMe. An enzyme carboxylate group is thought to be responsible for nucleophilic attack on the cyclopropane ring leading to irreversible inactivation.
The actions of a red tide toxin from Ptychodiscus brevis on single sodium channels in mammalian neuroblastoma cells
Authors: Sheridan Robert E. , Adler Michael
Published: July 2002

The actions of brevetoxin (PbTX‐3) were studied on single, voltage‐dependent sodium channels and whole‐cell currents from the neuroblastoma × glioma cell line NG108‐15. Purified PbTX‐3 shifted the activation of sodium channels to membrane potentials negative to normal. PbTX‐3 did not alter the single‐channel mean open lifetime, suggesting that the toxin does not change the rate of sodium channel inactivation from the open state. There was also no change in single‐channel conductance. These results indicate that brevetoxin increases sodium current at rest by shifting the voltage dependence of channel activation and that the resulting depolarization is limited by channel inactivation.
Regulation of hepatic expression of IGF I and fetal IGF binding protein mRNA in streptozotocin‐diabetic rats
Authors: Böni-Schnetzler M. , Binz K. , Mary J.-L. , Schmid C. , Schwander J. , Froesch E.R.
Published: July 2002

Hepatic mRNA levels of insulin‐like growth factor I (IGF I) and of the fetal, nonglycosylated 32 kDa IGF‐binding protein (BP) were analysed in diabetic, diabetic insulin‐ and IGF I‐treated rats as well as in age‐matched, healthy control animals. IGF I mRNA levels are reduced in diabetic rats and increased by insulin treatment. In contrast, the infusion of IGF I does not significantly upregulate IGF I mRNA levels. Fetal IGF BP mRNA expression is very low in healthy control animals, but high levels are found in diabetic rats. Insulin therapy lowers fetal IGF BP mRNA levels, whereas IGF I has no efrect. We propose that insulin is a major regulator of the 32 kDa IGF BP levels in adult rats.
Pathobiochemistry of CO poisoning
Authors: Young Lawrence J. , Caughey Winslow S.
Published: July 2002

A hypothesis for the pathobiochemical mechanism of CO poisoning, amenable to in vivo testing with optical reflectance spectrophotometry, is presented. It differs from the classical formulation in which loss of cytochrome c oxidase function is attributed entirely to O2 depletion, by including ligation and oxygenation of CO as essential components of the inhibitory process.
Amino acid sequence of the Ca2+‐triggered luciferin binding protein of Renilla reniformis
Authors: Kumar Santosh , Harrylock Maria , Walsh Kenneth A. , Cormier Milton J. , Charbonneau Harry
Published: July 2002

The complete amino acid sequence of the Ca2+‐triggered luciferin binding protein (LBP) of Renilla reniformis has been determined. The apoprotein has an unblocked amino terminus and contains 184 residues with a calculated M
r of 20541. LBP is a member of the EF‐hand superfamily of Ca2+‐binding proteins and bears three predicted EF‐hand domains. The sequence and organization of EF‐hand domains are similar to those of the Ca2+‐dependent photoprotein, aequorin.
Replacement of threonine residues by serine and alanine in a phosphorylatable heavy chain fragment of Dictyostelium myosin II
Authors: Lück-Vielmetter Dorothea , Schleicher Michael , Grabatin Barbara , Wippler Jürgen , Gerisch Günther
Published: July 2002

The target sites of soluble myosin heavy chain kinases partially purified from growth phase or aggregation competent cells of Dictyostelium discoideum were identified by the use of normal and mutated fragments of the myosin heavy chain. The kinases from both developmental stages phosphorylated two previously established threonine residues, as well as an additional one. The newly identified site is located within the putative core region of the coiled‐coil formed by the myosin tail. A lysine following the phosphorylated threonine residue is the only common feature of the sequences around these sites. The kinases, which specifically phosphorylate threonine residues in wild‐type myosin, did accept serine if it was in the right structural context.
Cloning and structure of cDNA encoding α‐latrotoxin from black widow spider venom
Authors: Kiyatkin N.I. , Dulubova I.E. , Chekhovskaya I.A. , Grishin E.V.
Published: July 2002

cDNA encoding the putative α‐latrotoxin precursor was isolated from spider venom glands cDNA library and sequenced. The cDNA contained the 4203 base‐pair open reading frame corresponding to the 156 855‐Da protein composed of 1401 amino acids. Computer analysis of the deduced primary structure revealed the presence of various internal imperfect repeats mainly in its central and C‐tenninal regions.
Porcine leukocyte 5‐ and 12‐lipoxygenases are iron enzymes
Authors: Kroneck Peter M.H. , Cucurou Christophe , Ullrich Volker , Ueda Natsuo , Suzuki Hiroshi , Yoshimoto Tanihiro , Matsuda Satoshi , Yamamoto Shozo
Published: July 2002

5‐ and 12‐lipoxygenases isolated from porcine leukocytes were investigated by electron paramagnetic resonance at X‐band and atomic absorption spectroscopy. For comparison potato 5‐lipoxygenase was studied under identical experimental conditions. All three lipoxygenases contained between 0.7 and 0.9 Fe atoms/enzyme molecule. As isolated, both mammalian enzymes exhibited a characteristic EPR signal at low magnetic field with a maximum at g =5.20 indicative of a high‐spin ferric iron center. The signal was not affected by the oxidants 12‐hydroperoxyeicosatetraenoic acid or arachidonic acid, nor was it affected by the reductant nordihydroguaiaretic acid. In the case of the potato enzyme an intense EPR signal with resonances at g =7.50, 6.39 and 5.84 was only observed after addition of an oxidant, such as 9‐hydroperoxyoctadecadienoic acid.
Strategies for expression of foreign genes in plants Potential use of engineered viruses
Authors: Joshi Rajiv L. , Joshi Vandana
Published: July 2002

Advances in gene transfer techniques for higher plants have already permitted important achievements towards crop protection and improvement using recombinant DNA technology. Besides plant genetic engineering, the possible use of plant viruses to express foreign genes could be of considerable interest to plant biotechnology. However, insuring containment of engineered viruses for environmental use is an important safety issue that must be addressed.
Different regulation of collagen I gene transcription in three‐dimensional lattice cultures
Authors: Gillery Philippe , Leperre Armelle , Coustry Françoise , Maquart François-Xavier , Borel Jacques-Paul
Published: July 2002

Human skin fibroblasts were cultivated in confluent monolayers, retracting collagen lattices, retracting fibrin lattices and non‐retracting fibrin lattices and the expression of messenger RNA specific for the α1 chain of type I procollagen comparatively studied by Northern blot and dot blot hybridization. Two factors contribute to the lower level of procollagen messenger RNA in collagen lattices the retraction and the nature of the fibrillar protein that constitutes the lattices. Fibrin lattices, when they do not retract, make as much collagen and procollagen mRNA as monolayer confluent cells.
The clearance of apoptotic cells in the liver is mediated by the asialoglycoprotein receptor
Authors: Dini Luciana , Autuori Francesco , Lentini Alessandro , Oliverio Serafina , Piacentini Mauro
Published: July 2002

Apoptosing cells are actively phagocytosed in parenchymal tissues, thus preventing the inflammatory reaction which could derive from their slow uncontrolled degradation. The molecular mechanisms by which an apoptotic cell is recognized and taken up are largely unknown. We propose that the recognition of apoptotic hepatocytes is mediated by the sugar recognition systems of the liver, particularly the asialoglycoprotein receptor (ASGP‐R). The results presented here demonstrate the participation of ASGP‐R in the removal of apoptotic parenchymal cells, and indicate a new perspective for the understanding of its physiological role.
Evidence that dopachrome tautomerase is a ferrous iron‐binding glycoprotein
Authors: Chakraborty Ashok K. , Orlow Seth J. , Pawelek John M.
Published: July 2002

Dopachrome tautomerase (DT) (EC 5.3.2.3) is a melanocyte‐specific, membrane‐associated, heat‐labile, non‐dialyzable, protease‐sensitive factor which catalyzes the isomeric rearrangement of dopachrome to 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA), apparently through a tautomerization reaction. Metal ions such as Cu, Ni, Co, Zn, Mn, Ca, Al, and Fe can also catalyze the dopachrome/DHICA isomerization. How is the reaction regulated in vivo? An attractive possibility would be that DT is a metalloenzyme. Here we present evidence that this may indeed be the case. Purified preparations or DT and tyrosinase, obtained from Cloudman S91 mouse melanoma cells. were assayed in the presence of a variety of metal chelators including EDTA (predominantly Ca and Mg). EGTA (predominantly Ca), phenylthiourea (PTU) (predominantly Cu), 2,2′‐dipyridyl (predominantly Fe): 1,10‐phenanthroline (predominantly Fe), and 2,3‐dihydroxybenzoic acid (predominantly Fe). In addition. DT activity was assayed in the presence of two non‐chelating structural analogs of 1,10‐phenanthroline, Results were as follows: (i) iron chelators inhibited DT activity with no effects on tyrosinase activity; (ii) inhibition by the chelators was reversible with the addition of Ferrous iron; (iii) 1,10‐phenanthroline pre‐complexed to Ferrous iron was not inhibitory to DT: (iv) non‐chelating analogs of phenanthroline were not inhibitory to DT; (v) PTU was inhibitory to tyrosinase but not DT; (vi) Ca2+ and Mg2+ chelators had little effect on either enzyme activity. Finally, studies with glycosylation inhibitors, glycosylase enzymes, and immobilized lectins, indicated that DT is a glycoprotein. The results suggest that DT is a metal‐containing glycosylated enzyme, possibly with Ferrous iron at its catalytic center.
Synthesis of the proline analogue [2,3‐3H]azetidine‐2‐carboxylic acid Uptake and incorporation in Arabidopsis thaliana and Escherichia coli
Authors: Verbruggen Nathalie , van Montagu Marc , Messens Eric
Published: July 2002

Azetidine‐2‐carboxylic acid, the 4‐membered ring noranalogue or proline, is regularly used in the study or proline metabolism as well as the study of protein conformation. We prepared ??‐[2,3‐3H]azetidine‐2‐carboxylic acid with an optimized 10% yield from commercially available 4‐amino‐[2,3‐3H]butyric acid. Purification was performed by fast‐protein liquid chromatography. The biological activity was checked in both Arabidopsis thaliana and Escherichia coli. The obtained specific activity or 10 mCl/mmol was sufficient for most uptake and incorporation studies.
Myoglobin protects against endothelial cell membrane damage associated with hydrogen peroxide or xanthine/xanthine oxidase
Authors: Yang Weidong , de Bono David
Published: July 2002

Oxymyoglobin at ‘physiological’ concentrations of 20–100 micromolar protected cultured endothelial cells from damage by xanthine/xanthine oxidase or by hydrogen peroxide. Metmyoglobin also provided a degree of protection, but apomyoglobin was ineffective. Protection was enhanced in the presence of ascorbate (0.01–1 mM). Myoglobin may have a physiological role in the protection of muscular tissue from isehaemia/reperfusion‐induced damage.
Insertion of MOM22 into the mitochondrial outer membrane strictly depends on surface receptors
Authors: Keil Petra , Pfanner Nikolaus
Published: July 2002

Targeting of preproteins to mitochondria and their translocation across the outer membrane are mediated by the mitochondrial receptor complex. This protein complex contains the import receptors MOM19 and MOM72 and the general insertion pore GIP. All seven components of the receptor complex are synthesized in the cytosol and thus have to be targeted to the mitochondria themselves. Here we investigated the import pathway of the precursor of MOM22 into the outer membrane. In contrast to other mitochondrial preproteins studied so far, the import of MOM22 absolutely depended on the presence of surface receptors. In fact, both receptors MOM19 and MOM72 were involved in its import pathway. The targeting of MOM22 to mitochondria is thus highly specific and controlled.
Adaptability of nonnatural aromatic amino acids to the active center of the E. coli ribosomal A site
Authors: Hohsaka Takahiro , Sato Ken , Sisido Masahiko , Takai Kazuyuki , Yokoyama Shigeyuki
Published: July 2002

3'‐N‐Aminoacyl analogs of puromycin with nonnatural aromatic amino acids were synthesized and their inhibitory activity in E.coli in vitro protein synthesizing system was evaluated. The analogs with l‐2‐naphthylalanine, l‐p‐biphenylalanine, l‐2‐anthrylalanine and trans‐l‐p‐phenylazophenylalanine were found to inhibit the protein synthesis with high efficiency. The inhibition suggests that these nonnatural amino acids are accepted by the active center of the E. coli ribosomal A site. A model for the adaptability of nonnatural aromatic amino acids to the active center is proposed.
Genetic polymorphism of cytochrome P4502E1 in a Swedish population
Authors: Persson Irene , Johansson Inger , Bergung Helene , Dahl Marja-Liisa , Seidegård Janeric , Rylander Ragnar , Rannug Agneta , Högberg Johan , Sundberg Magnus Ingelman
Published: July 2002

Genetic polymorphism of CYP2E1 was investigated among 195 Swedish patients with lung cancer and 206 controls. Three different polymorphic sites were found, all in introns, using RFLP and the restriction enzymes DraI, RsaI and TaqI. The frequencies of the rare alleles were 0.08–0.18 and much lower than previously described among Japanese. No significant difference in distribution of the polymorphic alleles between controls and lung cancer patients was evident, in contrast to results of a previous Japanese study. However, examination of a polymorphic site in the 5'‐flanking region, within a putative binding motif for the hepatic transcription factor HNF‐1, revealed a significantly less frequent distribution of the mutated allele (c2) among the lung cancer patients as compared to controls. It is concluded that major interethnic differences exist in the genetic polymorphism of CYP2E1 and that people carrying the c2 allele might be at lower risk for developing lung cancer.
Purification of the β product encoded by the Streptococcus pyogenes plasmid pSM19035
Authors: Rojo Fernando , Weise Frank , Alonso Juan C.
Published: July 2002

Genetic evidence suggests that the gene β product of Streptococcus pyogenes plasmid pSM19035 is required for converting plasmid multimers into monomers. The β protein was purified from cells overexpressing the cloned gene. N‐terminal protein sequence analysis demonstrated that the purified protein had the predicted sequence, except that the N‐terminal initiator methionine was not present. Native β protein consists of a dimer of two identical subunits with a molecular mass of 23.8 kDa (25 kDa in SDS‐PAGE). The β protein (isoelectric point of 9.7) binds specifically to a DNA fragment (312 bp in length) which contains the promoter region of the orf α‐gene β operon and two regions (sites I and II) that show dyad axes of symmetry. It is proposed that protein β binds to sites I and II to mediate resolution of plasmid oligomers.
The melanocortin (MC3) receptor from rat hypothalamus: Photoaffinity labelling and binding of alanine‐substituted α‐MSH analogues
Authors: Sahm U.G. , Qarawi M.A. , Olivier G.W.J. , Ahmed A.R.H. , Branch S.K. , Moss S.H. , Pouton C.W.
Published: July 2002

Membrane preparations of cells expressing the cloned rat hypothalamus melanocortin receptor, MC3, have been photoaffinity labelled using a radiolabelled photoreactive analogue of α‐MSH, [125I‐Tyr2,Nle4,d‐Phe7,ATB‐Lys11]α‐MSH.SDS‐PAGE followed by autoradiography showed a single band at 53–56 kDA for the native receptor of 35 kDA after deglycosylated with PNGase F, consistent with the predicted cDNA. Receptor binding studies with α‐MSH, γ‐MSH and [Nle4,d‐Phe7]α‐MSH established that α‐MSH and γ‐MSH had similar affinities while [Nle4,d‐Phe7]α‐MSH bound 100 times more strongly. These results suggest that the receptor recognises the conserved ‘core sequence’ (‐Met‐Glu/Gly‐His‐Phe‐Arg‐Trp‐) of MSH/ACTH peptides. The binding affinities of alanine‐substituted analogues of α‐MSH were determined to investigate the role of individual residues in ligand—receptor interactions. While in the terminal regions only the replacement of Tyr2 reduced the affinity of the peptide, replacement of Met4, Phe7, Arg8 and Trp9 within the peptide core led to a significant loss of affinity. Glu5 appeared unimportant for receptor recognition.
Ovarian 3,β‐hydroxysteroid dehydrogenase/Δ5‐4‐isomerase of rainbow trout: Its cDNA cloning and properties of the enzyme expressed in a mammalian cell
Authors: Sakai Noriyoshi , Tanaka Minoru , Takahashi Mika , Fukada Sachiko , Ian Mason J. , Nagahama Yoshitaka
Published: July 2002

A cDNA clone encoding 3β‐hydroxysteroid dehydrogenase/Δ5‐4‐isomerase (3β‐HSD) was isolated from a cDNA library of rainbow trout ovarian thecal cells. Southern hybridization analysis of trout genomic DNA with the cDNA suggested the presence of a single gene encoding 3β‐HSD in the rainbow trout showing a total genomic size of less than 4 kilobases (kb). The cDNA hybridized to a single species of mRNA isolated from rainbow trout ovaries; the 1.4 kb transcripts were most abundant in trout ovaries during the later stages of oogenesis. The trout 3β‐HSD expressed in non‐steroidogenic monkey kidney tumor (COS‐1) cells showed a unique enzymatic 3β‐HSD activity. Dehydroepiandrosterone was a more favoral substrate of the trout 3β‐HSD than 17α‐hydroxypregnenolone. Interestingly, the trout 3β‐HSD expressed in COS‐1 cells exhibited minimal ability to convert pregnenolone to progesterone.
Molecular cloning and expression of a prostaglandin E2 receptor of the EP3β subtype from rat hepatocytes
Authors: Neuschäfer-Rube Frank , DeVries Christa , Hänecke Kristina , Jungermann Kurt , Püschel Gerhard P.
Published: July 2002

Rat hepatocytes have previously been reported to possess prostaglandin E2 receptors of the EP3‐type (EP3‐receptors) that inhibit glucagon‐stimulated glycogenolysis by decreasing cAMP. Here, the isolation of a functional EP3β receptor cDNA clone from a rat hepatocyte cDNA library is reported. This clone can be translated into a 362‐amino‐acid protein, that displays over 95% homology to the EP3β receptor from mouse mastocytoma. The amino‐ and carboxy‐terminal region of the protein are least conserved. Transiently transfected HEK 293 cells expressed a single binding site for PGE2 with an apparent K
d of 15 nM. PGE2 > PGF2α > PGD2 competed for [3H]PGE2 binding sites as did the EP3 receptor agonists M&B 28767 = sulprostone > misoprostol but not the EP1 receptor antagonist SC 19220. In stably transfected CHO cells M&B 28767 > sulprostone = PGE2 > misoprostol > PGF2α inhibited the forskolin‐elicited cAMP formation. Thus, the characteristics of the EP3β receptor of rat hepatocytes closely resemble those of the EP3β receptor of mouse mastocytoma.
Pore‐forming peptide of Entamoeba histolytica significance of positively charged amino acid residues for its mode of action
Authors: Andrä Jörg , Leippe Matthias
Published: July 2002

Amoebapore is a 77‐residue pore‐forming peptide from Entamoeba histolytica with antibacterial and cytolytic properties. It contains eight lysine residues and one histidine residue. Chemical modifications of amoebapore with various reagents affecting either both types of cationic residues or lysine and histidine residues separately resulted in virtually complete loss of pore‐forming activity. The activity was restored by reversal of modifications. Whereas amoebapore was no longer capable of binding to phospholipid vesicles when its lysine residues were modified, the modification of the single histidine primarily affected oligomerization of the peptide upon membrane association.
The effects of antidepressant treatment on sleep disturbances in depression
Authors: Kerkhofs M , Mendlewicz J
Published: July 2002

Sleep problems are classical complaints in depressed patients. Polygraphie recordings performed in drug free depressed patients during an acute episode of the illness have allowed description of sleep continuity, architecture and REM sleep disturbances. While the specificity of these abnormalities is not fully established, these perturbations appear to be core symptoms of the depressive episode. Antidepressants drugs, whatever their pharmacological profile have been shown to suppress or at least to reduce REM sleep. However, the effects of these drugs on sleep continuity and architecture seem to be related to their specific pharmacological action.
Drug‐induced hypomethylation of a posttranscriptionally silenced transgene locus of tobacco leads to partial release of silencing
Authors: Kovařı́k Ales , Van Houdt Helena , Holý Antonı́n , Depicker Anna
Published: July 2002

The effect of DNA methylation upon posttranscriptional gene silencing (PTGS) has been investigated in transgenic tobacco lines showing PTGS and methylation of the neomycin phosphotransferase II (nptII) reporter genes. Application of the hypomethylation drugs dihydroxypropyladenine or 5‐azacytidine resulted in approximately 30% reduced methylation of cytosines located in a non‐symmetrical context in the 3′ untranslated region of the nptII transgenes. The hypomethylation was accompanied by up to 12‐fold increase in NPTII protein levels, suggesting that methylation of non‐symmetrical motifs may account for an increased degree of PTGS. Models for the possible role of DNA methylation in PTGS are discussed.
Characterization of a C‐terminal‐type kinesin‐related protein fromDictyostelium discoideum
Authors: Iwai Sosuke , Suyama Eigo , Adachi Hiroyuki , Sutoh Kazuo
Published: July 2002

We have determined the full sequence of K2, a kinesin‐related protein (KRP) inDictyostelium discoideum. Sequence homology and domain organization placed K2 in the ncd/Kar3 subfamily of the C‐terminal‐type KRPs. Bacterially expressed, truncated K2 showed ATP‐dependent binding to microtubules and microtubule‐stimulated ATPase activity. K2‐null cells grew and developed normally, suggesting overlapping functions of K2 with other microtubule motor(s). Overexpression of K2 caused partial mitotic arrest. Green fluorescent protein‐tagged full‐length K2 localized in the nucleus at the interphase and on the mitotic spindle during mitosis. These results suggest that K2 is a microtubule‐dependent motor which may play some roles in mitotic spindles.
Interaction of viper venom serine peptidases with thrombin receptors on human platelets
Authors: Santos Beatriz F. , Serrano Solange M.T. , Kuliopulos Athan , Niewiarowski Stefan
Published: July 2002

The serine peptidases, thrombocytin and PA‐BJ, isolated from the venom of Bothrops atrox and Bothrops jararaca, respectively, induce platelet aggregation and granule secretion without clotting fibrinogen. The specific platelet aggregation activity of each enzyme was about 15 times lower than that of thrombin. This activity was blocked by monoclonal antibodies recognizing protease activated receptor 1 (PAR1) and by heparin, but not by hirudin nor thrombomodulin. Both enzymes induced calcium mobilization in platelets and desensitized platelets to the action of thrombin and the SFLLRN peptide. We compared the effect of thrombin, PA‐BJ, and thrombocytin on the degradation of the soluble N‐terminal domain of the PAR1 receptor. The major cleavage site by thrombin and both viper enzymes was Arg41–Ser42. In addition, a rapid cleavage of the peptide bond at Arg46–Asn47 by the viper enzymes was observed, resulting in the inactivation of the tethered ligand. PA‐BJ and thrombocytin both cleaved at 41–42 and 46–47 peptide bonds, and fragment 42–103 disappeared rapidly. Both viper enzymes caused calcium mobilization in fibroblasts transfected with PAR4 and desensitized these cells to the thrombin action. In conclusion, both PAR1 and PAR4 mediate the effect of viper venom serine peptidases on platelets.
Interactions of bile salt micelles and colipase studied through intermolecular nOes
Authors: Dominguez Cyril , Sebban-Kreuzer Corinne , Bornet Olivier , Kerfelec Brigitte , Chapus Catherine , Guerlesquin Françoise
Published: July 2002

Colipase is a small protein (10 kDa), which acts as a protein cofactor for the pancreatic lipase. Various models of the activated ternary complex (lipase–colipase–bile salt micelles) have been proposed using detergent micelles, but no structural information has been established with bile salt micelles. We have investigated the organization of sodium taurodeoxycholate (NaTDC) micelles and their interactions with pig and horse colipases by homonuclear nuclear magnetic resonance (NMR) spectroscopy. The NMR data supply evidence that the folding of horse colipase is similar to that already described for pig colipase. Intermolecular nuclear Overhauser effects have shown that two conserved aromatic residues interact with NaTDC micelles.
Phosphorylation of vimentin head domain inhibits interaction with the carboxyl‐terminal end of α‐helical rod domain studied by surface plasmon resonance measurements
Authors: Gohara Rumi , Tang Dahai , Inada Hiroyasu , Inagaki Masaki , Takasaki Yozo , Ando Shoji
Published: July 2002

The amino‐terminal head domain of vimentin is the target site for several protein kinases and phosphorylation induces disassembly of the vimentin intermediate filaments in vivo and in vitro. To better understand molecular mechanisms involved in phosphorylation‐dependent disassembly, we examined domain interactions involving the head domain and the effect of phosphorylation on the interaction, using surface plasmon resonance. We observed that the head domain binds to the carboxyl‐terminal helix 2B in the rod domain, under physiological ionic strength. This interaction was interfered with by A‐kinase phosphorylation of the head domain. Deletion of the carboxyl‐terminal 20 amino acids of helix 2B resulted in loss of the interaction. Furthermore, peptide representing the carboxyl‐terminal 20 residues of helix 2B had a substantial affinity with the head domain but not with the phosphorylated one. These findings support the idea that the interaction between the head domain and the last 20 residues of helix 2B is essential for association of vimentin tetramers into the intermediate filaments and that the phosphorylation‐dependent disassembly is the result of loss of the interaction.
Mutations in the interdomain linker region of DnaK abolish the chaperone action of the DnaK/DnaJ/GrpE system
Authors: Han Wanjiang , Christen Philipp
Published: July 2002

Hsp70s assist the folding of proteins in an ATP‐dependent manner. DnaK, the Hsp70 of Escherichia coli, acts in concert with its co‐chaperones DnaJ and GrpE. Amino acid substitutions (D388R and L391S/L392G) in the linker region between the ATPase and substrate‐binding domain did not affect the functional domain coupling and oligomerization of DnaK. The intrinsic ATPase activity was enhanced up to 10‐fold. However, the ATPase activity of DnaK L391S/L392G, if stimulated by DnaJ plus protein substrate, was five times lower than that of wild‐type DnaK and DnaK D388R. This defect correlated with the complete loss of chaperone action in luciferase refolding. Apparently, the conserved leucine residues in the linker mediate the synergistic effects of DnaJ and protein substrate on ATPase activity, a function which might be essential for chaperone action.
Identification of novel mast cell genes by serial analysis of gene expression in cord blood‐derived mast cells
Authors: Kuramasu Atsuo , Kubota Yasuhiko , Matsumoto Kenji , Nakajima Toshiharu , Sun Xu-Ming , Watanabe Takehiko , Saito Hirohisa , Ohtsu Hiroshi
Published: July 2002

The gene expression profile of human cord blood‐derived mast cells (MCs) was investigated using serial analysis of gene expression (SAGE). A total of 22 914 tags, representing 9181 unique transcripts, were sequenced. By selecting tags that were detected more frequently in MCs than in other tissues, genes characteristic of MCs were enriched. Reverse transcription‐PCR and the high‐density oligonucleotide array hybridization confirmed the validity of our SAGE result. About 70% of the selected genes were previously uncharacterized. Northern blot analysis showed the MC‐specific expression of selected genes. This inventory will be useful to identify novel genes with important functions in MCs.
Prenylation of Rab GTPases: molecular mechanisms and involvement in genetic disease
Authors: Pereira-Leal José B , Hume Alistair N , Seabra Miguel C
Published: July 2002

Small GTPases of the Rab family regulate membrane transport pathways. More than 50 mammalian Rab proteins are known, many with transport step‐specific localisation. Rabs must associate with cellular membranes for activity and membrane attachment is mediated by prenyl (geranylgeranyl) post‐translational modification. Mutations in genes encoding proteins essential for the geranylgeranylation reaction, Rab escort protein and Rab geranylgeranyl transferase, underlie genetic diseases. Choroideremia patients have loss of function mutations in REP1 and the murine Hermansky–Pudlak syndrome model gunmetal possesses a splice‐site mutation in the α‐subunit of RGGT. Here we discuss recent insights into Rab prenylation and advances towards our understanding of both diseases.
Towards a mechanism of AMP‐substrate inhibition in adenylate kinase from Escherichia coli
Authors: Sinev Michael A. , Sineva Elena V. , Ittah Varda , Haas Elisha
Published: July 2002

Crystallographic studies on adenylate kinase (AK) suggest that binding of ATP causes the LID domain of the enzyme to close over the ATP molecule (Schlauderer et al. (1996) J. Mol. Biol. 256, 223–227). The method of time‐resolved fluorescence resonance energy transfer was applied to study the proposed structural change in AK from Escherichia coli. Two active derivatives of the (C77S, A73C, V142C)‐AK mutant containing the excitation energy donor attached to one of the two cysteine residues and the acceptor attached to the other cysteine were prepared to monitor displacements of the LID domain in response to substrate binding. Binding of either ATP or AMP was accompanied by a ∼ 9 A decrease in the interprobe distances suggesting LID domain closure. Closure of the LID domain in response to AMP binding may be a possible reason for the strong AMP‐substrate inhibition known for E. coli AK.
Removal of substrate inhibition in a lactate dehydrogenase from human muscle by a single residue change
Authors: Eszes Csilla M. , Sessions Richard B. , Clarke Anthony R. , Moreton Kathleen M. , Holbrook J.John
Published: July 2002

High concentrations of ketoacid substrates inhibit most natural hydroxyacid dehydrogenases due to the formation of an abortive enzyme‐NAD+‐ketoacid complex. It was postulated that this substrate inhibition could be eliminated from lactate dehydrogenases if the rate of NAD+ dissociation could be increased. An analysis of the crystal structure of mammalian LDHs showed that the amide of the nicotinamide cofactor formed a water‐bridged hydrogen bond to S163. The LDH of Plasmodium falciparum is not inhibited by its substrate and, uniquely, in this enzyme the serine is replaced by a leucine. In the S163L mutant of human LDH‐M4 pyruvate inhibition is, indeed, abolished and the enzyme retains high activity. However, the major contribution to this effect comes from a weakening of the interaction of pyruvate with the enzyme‐coenzyme complex.
Substitution of just five nucleotides at and around the transcription start site of rat β‐actin promoter is sufficient to render the resulting transcript a subject for translational control
Authors: Biberman Yael , Meyuhas Oded
Published: July 2002

Vertebrate mRNAs with a 5′ terminal oligopyrimidine tract (5′ TOP), including those encoding ribosomal proteins and elongation factors, are candidates for translational control in a growth‐dependent fashion. The present study was designed to determine the minimal cis‐regulatory element involved in this mode of regulation. We selected rat β‐actin mRNA, a typical translationally uncontrolled transcript, as a subject for gain‐of‐function analysis. Mutations at and around its cap site leading to the formation of a 7 pyrimidines long 5′ TOP render the resulting transcript translationally repressed upon growth arrest of lymphosarcoma cells. In contrast, growth‐dependent translational control of this mRNA in fibroblasts requires, in addition, a GC motif downstream of the 5′ TOP. A similar motif is present in all ribosomal prtein mRNAs shown to be translationally controlled.
Movement of the 3′‐end of tRNA through the peptidyl transferase centre and its inhibition by antibiotics
Authors: Kirillov Stanislav , Porse Bo T. , Vester Birte , Woolley Paul , Garrett Roger A.
Published: July 2002

Determining how antibiotics inhibit ribosomal activity requires a detailed understanding of the interactions and relative movement of tRNA, mRNA and the ribosome. Recent models for the formation of hybrid tRNA binding sites during the elongation cycle have provided a basis for re‐evaluating earlier experimental data and, especially, those relevant to substrate movements through the peptidyl transferase centre. With the exception of deacylated tRNA, which binds at the E‐site, ribosomal interactions of the 3′‐ends of the tRNA substrates generate only a small part of the total free energy of tRNA‐ribosome binding. Nevertheless, these relatively weak interactions determine the unidirectional movement of tRNAs through the ribosome and, moreover, they appear to be particularly susceptible to perturbation by antibiotics. Here we summarise current ideas relating particularly to the movement of the 3′‐ends of tRNA through the ribosome and consider possible inhibitory mechanisms of the peptidyl transferase antibiotics.
Testosterone induces Ca2+ influx via non‐genomic surface receptors in activated T cells
Authors: Benten W.Peter M , Lieberherr Michèle , Sekeris Constantin E , Wunderlich Frank
Published: July 2002

Using the Fura‐2 method we investigated a possible direct action of testosterone on cytosolic free calcium of splenic T cells isolated from female C57BL/10 mice. Testosterone at physiological concentrations of 1–10 nM induces an increase in [Ca2+]i within seconds, which is due to Ca2+ influx and not to Ca2+ release from intracellular stores. In contrast, estradiol induces both Ca2+ influx and Ca2+ release. The testosterone‐induced Ca2+ influx is mediated by Ni2+‐blockable channels and is not inhibited by cyproterone, a blocker of the classical androgen receptor. Ca2+ influx can also be induced by testosterone conjugated to BSA which is impermeable to the plasma membrane. These data indicate a novel mode of direct action of testosterone on T cells which is not mediated through the classical androgen receptor response, but through unconventional plasma membrane receptors.
Analysis of phosphofructokinase subunits and isozymes in ascites tumor cells and its original tissue, murine mammary gland
Authors: Sánchez-Martı́nez Cristina , J. Aragón Juan
Published: July 2002

Phosphofructokinase (PFK) subunits and isozymes have been examined in ascites tumor cells and murine mammary gland, the tissue from where this tumor originated. Ascites tumor was found to contain predominantly the C‐type subunit, whereas the L‐type subunit was more abundant in mammary gland. An altered M‐type subunit of lower electrophoretic mobility was found in both cell types and no M4 homotetramers were detected in either of them. Characteristic regulatory properties of ascites tumor PFK, i.e. insensitivity to fructose‐1,6‐P2 activation and inhibition by P‐enolpyruvate, were also observed in the mammary gland isozyme. The nature of these properties and the contribution of the distinct subunit types to fructose‐1,6‐P2 activation are discussed.
Properties of the extracellular calcium binding module of the proteoglycan testican
Authors: Kohfeldt Eddie , Maurer Patrik , Vannahme Christian , Timpl Rupert
Published: July 2002

The extracellular calcium‐binding (EC) module of human testican (115 residues) was obtained in native form by recombinant production in mammalian cell culture and thus shown to represent an independently folding domain. This module showed a large loss in α‐helix upon calcium depletion. Apparently only one of the two EF hands binds calcium, with a moderate affinity (K
d=68 μM) about 100‐fold lower than in the homologous BM‐40 protein. No clear evidence was obtained for collagen binding, indicating that EC modules found in different proteins may not share similar functions.
Modulation of membrane activity of amphipathic, antibacterial peptides by slight modifications of the hydrophobic moment
Authors: Wieprecht Torsten , Dathe Margitta , Krause Eberhard , Beyermann Michael , Maloy W.Lee , MacDonald Dorothy L , Bienert Michael
Published: July 2002

Starting from the sequences of magainin 2 analogs, peptides with slightly increased hydrophobic moment (μ) but retained other structural parameters were designed. Circular dichroism investigations revealed that all peptides adopt an α‐helical conformation when bound to phospholipid vesicles. Analogs with increased μ were considerably more active in permeabilizing vesicles mainly composed of zwitterionic lipid. In addition, the antibacterial and hemolytic activities of these analogs were enhanced. Correlation of permeabilization and binding indicated that the activity increase is predominantly caused by an increased membrane affinity of the peptides due to strengthened hydrophobic interactions.
The amyloidogenic potential of transthyretin variants correlates with their tendency to aggregate in solution
Authors: Quintas Alexandre , Saraiva M.J.M , Brito Rui M.M
Published: July 2002

Amyloid fibril formation and deposition are the basis for a wide range of diseases, including spongiform encephalopathies, Alzheimer's and familial amyloidotic polyneuropathies. However, the molecular mechanisms of amyloid formation are still poorly characterised. In certain forms of familial amyloidotic polyneuropathy (FAP), the amyloid fibrils are mostly constituted by variants of transthyretin (TTR). V30M‐TTR is the most frequent variant, and L55P‐TTR is the variant associated with the most aggressive form of amyloidosis. Here, we report gel filtration chromatography experiments to characterise the aggregation states of WT‐, V30M‐, L55P‐TTR and a non‐amyloidogenic variant, T119M‐TTR, in solution, at nearly physiological pH. These studies show that all four protein tetramers dissociate to monomer upon dilution, in the sub‐micromolar range, at pH 7.0. The amyloidogenic proteins V30M‐ and L55P‐TTR show a complex equilibrium between monomers, tetramers and high molecular weight aggregate species. These aggregates dissociate directly to monomer upon dilution. This study shows that the tendency to form aggregates among the four studied proteins correlates with their known amyloidogenic potential. Thus, the amyloidogenic mutations could perturb the structure and/or stability of the monomeric species leading initially to the formation of soluble aggregates and at a later stage to insoluble amyloid fibrils.
Micro‐injection of recombinant lysyl oxidase blocks oncogenic p21‐Ha‐Ras and progesterone effects on Xenopus laevis oocyte maturation
Authors: Di Donato Armando , Lacal Juan Carlos , Di Duca Marco , Giampuzzi Monia , Ghiggeri Gianmarco , Gusmano Rosanna
Published: July 2002

Previous evidence suggested an anti‐oncogenic role for lysyl oxidase, mainly in ras‐transformed cells. Here we prove that recombinant lysyl oxidase is actually able to antagonize p21‐Ha‐Ras‐induced Xenopus laevis oocyte maturation. Lysyl oxidase was also effective on progesterone‐dependent maturation, indicating a block lying downstream of Ras. Maturation induced by activated ‘maturation promoting factor’, normally triggered by progesterone, was also inhibited by lysyl oxidase. Finally, lysyl oxidase did not abolish p42Erk2 phosphorylation upon maturation triggering, suggesting a block downstream of Erk2. Further investigation showed that lysyl oxidase action depends on protein synthesis and is therefore probably mediated by a newly synthesized protein.
P1,P4‐diadenosine 5′ tetraphosphate induces nitric oxide release from bovine aortic endothelial cells
Authors: Hilderman Richard H , Christensen Eric F
Published: July 2002

Since the infusion of P1,P4‐diadenosine 5′ tetraphosphate (Ap4A) into animal models induces vasodilation [1, 2], the present study was performed to determine whether Ap4A induces the release of nitric oxide (NO) from endothelial cells. Ap4A induced NO release was 4.2‐fold greater than the amount of NO released under basal condition. Ap4A induced NO release was inhibited by N
G‐nitro‐l‐arginine (L‐NNA) and this inhibition was reversed by L‐Arg. In addition, EGTA inhibits Ap4A induced NO release. These data are consistent with Ap4A inducing the release of NO from endothelial cells through the activation of endothelial nitric oxide synthase.
The cloning and developmental regulation of murine diacylglycerol kinase ζ
Authors: Ding Li , McIntyre Thomas M , Zimmerman Guy A , Prescott Stephen M
Published: July 2002

Diacylglycerol kinases (DGKs) regulate the key signaling intermediates diacylglycerol (DAG) and phosphatidic acid (PA). We isolated cDNA clones of mouse diacylglycerol kinase ζ (mDGKζ) and found that it shares 88% identity at the nucleic acid level and 95.5% identity at the amino acid level with human DGKζ (hDGKζ). Murine DGKζ protein rose gradually during embryonic development, and was abundant in newborn and adult brains. By RNA whole‐mount in situ hybridization, mDGKζ was shown to be expressed in spinal ganglia and limb buds at low level in E11.5 embryos and at higher level in E12.5 embryos. In E13.5 embryos, DGKζ mRNA was highly expressed in vibrissa follicles, in spinal ganglia, and in the interdigital regions of the developing limbs. Northern blotting showed that DGKζ expression was limited to specific anatomical regions of the brain. Thus, the expression of DGKζ is regulated temporally and spatially during mammalian development and correlates with the development of sensory neurons and regions undergoing apoptosis.
Effect of chloroquine and leupeptin on intracellular accumulation of amyloid‐beta (Aβ) 1–42 peptide in a murine N9 microglial cell line
Authors: Chu Teresa , Tran Thuy , Yang Fusheng , Beech Walter , Cole Greg M , Frautschy Sally A
Published: July 2002

Murine N9 microglia accumulated Aβ from media containing 0.67 μM Aβ within 6 h. In N9 and in primary rat microglia, chloroquine, which disrupts lysosomal pH, increased Aβ‐induced accumulation of Aβ, particularly Aβ1–42. Leupeptin similarly enhanced Aβ accumulation. The scavenger receptor antagonist fucoidan did not affect acute chloroquine‐dependent Aβ1–42 accumulation, demonstrating uptake of non‐aggregated Aβ. After prolonged incubations, chloroquine enhanced Aβ multimer (8–12 kDa) accumulation, an effect inhibited by fucoidan. Disruptions of the lysosomal system enhance Aβ and its multimer formation. Despite negligible effects of fucoidan on initial Aβ uptake, chronic exposure inhibits multimer accumulation, demonstrating a role for scavenger receptor in multimer accumulation.
Kinetic properties and stereospecificity of the monomeric dUTPase from herpes simplex virus type 1
Authors: Bergman Anna-Carin , Nyman Per Olof , Larsson Gunilla
Published: July 2002

Kinetic properties of the monomeric enzyme dUTPase from herpes simplex virus type 1 (HSV) were investigated and compared to those previously determined for homotrimeric dUTPases of bacterial and retroviral origins. The HSV and Escherichia coli dUTPases are equally potent as catalysts towards the native substrate dUTP with a k
cat/K
M of about 107 M−1 s−1 and a K
M of 0.3 μM. However, the viral enzymes are less specific than the bacterial enzyme. The HSV and E. coli dUTPases show the same stereospecificity towards the racemic substrate analogue dUTPαS (2′‐deoxyuridine 5′‐(α‐thio)triphosphate), suggesting that they have identical reaction mechanisms.
The ras oncogene inhibits growth factor inducibility of early response genes, and promotes selectively expression of NGFI‐A in a PC12 cell line
Authors: Cosgaya José Miguel , Aranda Ana
Published: July 2002

Expression of oncogenic Ras in UR61 cells (a PC12 subclone) results in neuronal differentiation. We have observed that the oncoprotein selectively increased the levels of NGFI‐A transcripts, but was unable to induce NGFI‐B or c‐fos transcripts. In contrast, nerve growth factor (NGF) elicited a strong induction of the three immediate early genes (IEGs). Thus, activation of Ras alone is sufficient for the induction of NGFI‐A by NGF, whereas an additional pathway(s), besides Ras, is required for the stimulation of NGFI‐B and c‐fos gene expression. These results show that the acquisition of a neuronal phenotype does not correlate with induction of IEG expression. Additionally, Ras markedly reduces the response of the three genes to NGF and to other growth factors. This attenuation could reflect a negative regulatory mechanism acting on signalling pathways normally stimulated by growth factor receptors.