bluuebunny/crossref_metadata_2025 huggingface dataset:
Dermal glands of Xenopus laevis contain a polypeptide with a highly repetitive amino acid sequence
Authors: Gmachl Michael , Berger Hans , Thalhammer Josef , Kreil Günther
Published: July 2002

Mature dermal glands of Xenopus laevis contain storage granules with a characteristic ellipsoid shape. These granules contain, as a minor component, a heat‐stable, acidic polypeptide with an apparent molecular mass of 75 kDa. Using antibodies against this protein, positive clones were isolated from a cDNA expression library prepared from skin of X. leaevis. One of the cloned cDNAs encodes a pre‐protein with a typical signal sequence and a mature part of 396 amino acids. The protein contains 33 copies of the sequence Gly‐Gly/Glu‐(Ala‐Pro)2–4‐Ala‐Glu. Using the single‐letter code for the four predominant amino acids, we have termed this polypeptide the APEG protein. Near its carboxy‐terminus, one segment has been found with an amino acid sequence similar to that of spasmolytic polypeptide from porcine pancreas and to the human protein pS2.
Prochymosin activation by non‐aspartic proteinases
Authors: Stepanov V.M. , Lavrenova G.I. , Terent'eva E.Yu. , Khodova O.M.
Published: July 2002

Prochymosin can be converted into chymosin by an action of external proteinases. Thus, thermolysin at pH 5.05 converts calf prochymosin into active Phe‐chymosin, which is one amino acid longer than chymosin from the N‐terminus with a yield of 73%. Even better results were achieved with prochymosin activation by Legionella pneumophila metalloproteinase. Apparently the stretch of prochymosin polypeptide chain adjacent to the normally observed activation point becomes available for an attack by an external proteinase at pH 5.0–6.0. These data indicate that the intermolecular activation pathway might be of physiological importance.
Effects of propeptide deletion on human renin secretion from mouse pituitary AtT‐20 cells
Authors: Nagahama Masami , Nakayama Kazuhisa , Murakami Kazuo
Published: July 2002

To study the role of the N‐tenninal propeptide in the secretory process of renin, mouse pituitary AtT‐20 cells were transfected with expression plasmids of human preprorenin and a mutant deleted of its propeptide. The transfectant of the native construct secreted inactive prorenin and active renin, and renin secretion was stimulated by a secretagogue, 8‐Br‐cAMP. On the contrary, the transfectant of the deleted construct secreted only active renin, whose release was also stimulated by the secretagogue. The amount of renin molecule secreted from the latter transfectant was lower than that from the former one, although a significant amount of fully active renin could be produced. These results suggest that the propeptide plays an important role in the secretory process of renin, probably folding and/or stabilizing the renin molecule, but it does not contain the signal for intracellular sorting to target renin to secretory granules.
Quaternary structure of pigeon liver malic enzyme
Authors: Lee Hwei-Jen , Chang Gu-Gang
Published: July 2002

Pigeon liver malic enzyme (EC 1.1.1.40) has a double dimer quaternary structure. The NADP+ analogs, aminopyridine adenine dinucleotide phosphate and nicotinamide‐1.N
6‐ethenoadenosine dinucleotide phosphate, bind to the enzyme anti‐cooperatively. In the presence of non‐cooperative competing ligand NADP+ the binding parameter Hill coefficients of these analogues changed very little. Binding of L‐malate with enzyme‐AADP+ complex first enhanced then reduced the nucleotide fluorescence. Two L‐malate binding sites, with K
d values of 23–30 and 270–400 μM, respectively, for the tight and weak binding sites were postulated. A hybrid model between the sequential and pre‐existing asymmetrical models was proposed for the pigeon liver malic enzyme.
Zona pellucida‐binding of boar sperm acrosin is associated with the N‐terminal peptide of the acrosin B‐chain (heavy chain)
Authors: Töpfer-Petersen E. , Steinberger M. , Ebner von Eschenbach C. , Zucker A.
Published: July 2002

Recently, it has been shown that boar acrosin exhibits a carbohydrate‐binding activity with a specificity to fucose, by which it can bind to the oocyte zona pellucida. By limited autoproteolysis of a high‐molecular mass acrosin ( kDa), designated as α‐acrosin, a 15 kDa fragment was generated which interacts strongly with the porcine zona pellucida. Zona‐binding was demonstrated on protein blots and by the solid‐phase zonabinding assay utilizing biotinylated zona proteins. The zona‐binding peptide was isolated by reversed‐phase HPLC and analyzed for amino acid sequence. Its single N‐terminal sequence corresponded to that of the acrosin B‐chain (heavy chain). These data indicate that the zona‐binding properties of acrosin are associated with the N‐terminal peptide of the acrosin heavy chain.
The functional properties of full length and mutant chicken gizzard smooth muscle caldesmon expressed in Escherichia coli
Authors: Redwood C.S. , Marston S.B. , Bryan J. , Cross R.A. , Kendrick-Jones J.
Published: July 2002

Wild type chicken gizzard caldesmon (756 amino acids) was expressed in a T7 RNA polymerase‐based bacterial expression system at a yield of 1 mg pure caldesmon per litre bacterial culture. A mutant composed of amino acids 1‐578 was also constructed and expressed. The wild type and mutant caldesmon were purified and compared with native chicken gizzard caldesmon. Native and wild type expressed caldesmon were indistinguishable in assays for inhibition of actin‐tropomyosin activation of myosin ATPase, reversal of inhibition by Ca2+‐calmodulin and binding to actin, actin‐tropomyosin, Ca2+‐calmodulin, tropomyosin and myosin. The mutant missing the C‐terminal 178 amino acids had no inhibitory effect and did not bind to actin or Ca2+‐calmodulin. It bound to tropomyosin with a 5‐fold reduced affinity and to myosin with a greater than 10‐fold reduced affinity.
Localization of surface peptide from ribosomal protein L7 on 80 S ribosome by biotinylation
Authors: Lin Alan
Published: July 2002

A surface topography of ribosomal peptides on ribosome particles was conducted by using N',Hydroxysuccinimido‐biotin (NHS‐biotin) modification. All rat ribosomal proteins, except proteins L3 and L8, are biotinylated when the ribosome particle is the substrate. A surface peptide from protein L7 was determined from biotinylated ribosomes by high performance liquid chromatography and cyanogen bromide peptide mapping. It was found that only the tandem repeats of the NH2‐terminal segment of protein L7 are accessible to biotinylation. It is concluded that the NH2‐terminal‐end of protein L7 should be exposed on the surface of ribosomal particles.
Combination of Trp and Glu residues for recognition of mRNA cap structure Analysis of m7G base recognition site of human cap binding protein (IF‐4E) by site‐directed mutagenesis
Authors: Ueda Hitoshi , Iyo Hiromi , Doi Mitsunobu , Inoue Masatoshi , Ishida Toshimasa , Morioka Hiroshi , Tanaka Toshiki , Nishikawa Satoshi , Uesugi Seiichi
Published: July 2002

Four mutants of the human cap binding protein (hCBP), in which Trp‐102, Glu‐103, Asp‐104 or Glu‐105 was changed to the aliphatic Leu or Ala, were prepared, and their cap binding abilities were examined. Cap binding abilities of two mutants. W102L (Trp‐102→Leu) and E105A (Glu‐105→Ala), were significantly decreased in comparison with the wild‐type hCBP. This result suggest that Trp‐102 and Glu‐105 are both necessary for the cap binding, and the most probable binding mode with the m7G of cap structure is the combination of the stacking by Trp‐102 and the hydrogen‐bond pairing by Glu‐105, as was already proposed from the model studies.
Probing the secondary quinone (QB) environment in photosynthetic bacterial reaction centers by light‐induced FTIR difference spectroscopy
Authors: Breton J. , Berthomieu C. , Thibodeau D.L. , Nabedryk E.
Published: July 2002

The photoreduction of the secondary electron acceptor, QB, has been characterized by light‐induced Fourier transform infrared difference spectroscopy of Rb. sphaeroides and Rp. viridis reaction centers. The reaction centers were supplemented with ubiquinone (UQ10 or UQ0). The QB− state was generated either by continuous illumination at very low intensity or by single flash in the presence of redox compounds which rapidly reduce the photooxidized primary electron donor P+. This approach yields spectra free from P and P+ contributions making possible the study of the microenvironment of QB and QB−. Assignments are proposed for the CO vibration of QB− and tentatively for the C = O and C = C vibrations of QB.
Expression of human pituitary adenylate cyclase activating polypeptide (PACAP) cDNA in CHO cells and characterization of the products
Authors: Okazaki Kimitake , Kimura Chiharu , Kosaka Takuo , Watanabe Takuya , Ohkubo Shoichi , Ogi Kazuhiro , Kitada Chieko , Onda Haruo , Fujino Masahiko
Published: July 2002

cDNA encoding human PACAP precursor was expressed in non‐neuroendocrine Chinese hamster ovary cells, CHO‐K1. The cells were transfected with expression vector (pTS705) containing the human PACAP cDNA by electroporation. A cell line which produced more than 80 ng/ml of immunoreactive PACAP (ir‐PACAP) into the conditioned medium was established. RP‐HPLC analysis of culture medium of this established cell line exhibited the presence of two types of PACAP, i.e. PACAP38 and PACAP27. At the same time, it was also revealed that immunoreactive PACAP‐related peptide (ir‐PRP) was secreted into the cultured medium. The ir‐PACAPs were confirmed to have biological activities such as induction of cAMP and neurite outgrowth in rat pheochromocytoma PC12h cells.
Molecular cloning and primary structure of a protein phosphatase 2C isoform
Authors: Wenk Jutta , Trompeter Hans-Ingo , Pettrich Klaus-Günter , Cohen Patricia T.W. , Campbell David G. , Mieskes Gottfried
Published: July 2002

Complementary DNA encoding the isoform of protein phosphatase 2C, termed PP2C2, has been isolated. The cDNA predicts a protein of 390 amino acid residues with a molecular mass of 42,888 Da. The protein displays 76% identity to the PP2C1 isoform.
The complete primary structure of three isoforms of a boar sperm‐associated acrosin inhibitor
Authors: Jonáková Věra , Calvete Juan J. , Mann Karlheinz , Schäfer Wolfram , Schmid Erich R. , Töpfer-Petersen Edda
Published: July 2002

Acrosin inhibitors of seminal vesicle origin, after binding to their acceptor molecules on the anterior part of ejaculated sperm, are thought to be important capacitation factors, protecting zona binding sites during sperm uterine passage, and then dissociating to allow sperm binding to the zona pellucida of the oocyte. Each species so far tested possess an heterogeneous population of isoinhibitors which may display overlapping but not identical biological functions. Here we report the complete primary structure of three isoforms of a boar sperm‐associated acrosin inhibitor, whose sequences are 90% identical to the seminal plasma counterpart. Despite this high analogy, the differences between the sperm‐associated and the seminal plasma inhibitors may confer to them different physico‐chemical properties which are postulated to be of functional importance.
Ellipticity changes of the sarcoplasmic reticulum Ca2+‐ATPase induced by cation binding and phosphorylation
Authors: Girardet Jean-Luc , Dupont Yves
Published: July 2002

The sarcoplasmic reticulum (SR) Ca2+‐ATPase is a member of the ‘P‐type’ class of cation transport ATPases which form a covalent phosphorylated intermediate. It has been proposed that during ion transport, these proteins cyclically adopt two major enzymatic states E1 and E2, that are related to two essential conformations of the protein. By the use of especially sensitive circular dichroism (CD) instrumentation it is shown here that Ca2+ addition induces 5% or 2.5% increases in Ca2+‐ATPase ellipticity at 225 nm in the absence or in the presence of Mg2+, respectively. Furthermore, a 2% change in the same direction was observed when the enzyme was phosphorylated with Pi in the absence of Ca2+. These results suggest that the E1 ⇔ E2 transition and the E2‐P formation are associated with structural changes of the polypeptide backbone structure of the calcium pump protein.
Pregnancy hormones, estrogen and progesterone, prevent HIV‐1 synthesis in monocytes but not in lymphocytes
Authors: Bourinbaiar Aldar S. , Nagorny Raisa , Tan Xin
Published: July 2002

Increase in levels of estrogen and progesterone during pregnancy may affect intra‐uterine HIV‐1 injection through their effect on maternal immunocompetent cells. These hormones were examined for containing HIV‐1 production from ACH‐2 lymphocytes and U1 monocytes. Neither of the hormones has an effect on ACH‐2, but with U1, the physiological concentrations (0.1 μg‐0.1 ng) of progesterone and estrogen demonstrate significant inhibition of HIV‐1 release. Except for the highest dose of 1 μg/ml, the dose—response to progesterone and estrogen was not correlated with the negative influence on proliferation of both types of cells. The results suggest that in vivo low doses of female steroids may display specific antiviral activity in monocytes but not in lymphocytes.
Y2 receptor proteins for peptide YY and neuropeptide Y Characterization as N‐linked complex glycoproteins
Authors: Hansen Anette P. , Sheikh Søren P.
Published: July 2002

Affinity labeling using [125I‐Tyr36]PYY and homobifunctional affinity crosslinking reagents of the rabbit Y2 receptor for peptide YY(PYY) results in specifically labeled proteins of both Mr
=50,000 to 60,000 and Mr
=96,000 to 115,000 [1,2]. In this work the glycoprotoin nature of affinity labeled Y2 receptor proteins were investigated by enzymatic deglycosylation using neuraminidase, endoglycosidase F (endo F), N‐glycosidase F (PNGase F), and O‐glycanase treatment. Only N‐glycosidase F and neuraminidase increased the electrophoretic mobility of the radiolabeled receptor bands, whereas all other glycosidases did not. PNGase F treatment of both radiolabeled receptor bands electroeluted from gel slices reduced the apparent molecular mass of by 16–17 kDa units, that is Mr
=96,000 to 79,000 and Mr
=60,000 to 44,000, indicating removal of N‐linked oligosaccharide chains of similar size from both species. Neuraminidase treatment caused slight increases in the electrophoretic mobilities suggesting the presence of terminal sialic residues. It is concluded that the Y2 binding proteins are N‐linked complex (sialo)glycoproteins with a minimal core protein size of Mr
=44,000. Furthermore, based on this sensitivity pattern of the glycosidases, the Asn‐linked carbohydrate may be of the tri‐ or tetra‐antennary complex type containing terminal sialic acid residues.
Enhancement of epidermal growth factor (EGF) and insulin‐stimulated tyrosine phosphorylation of endogenous substrates by sodium selenate
Authors: Pillay T.S. , Makgoba M.W.
Published: July 2002

Sodium selenate stimulated tyrosine phosphorylation of the epidermal growth factor (EGF) receptor in A431 cells and enhanced the tyrosine phosphorylation of endogenous proteins in response to EGF in A431 cells and insulin in NIH 3T3 HIR3.5 cells. These effects occurred without changes in ligand binding, were not abolished by mercaptoethanol: in the case of the EGF receptor, and appeared distinct from the effects of vanadate. These results support a role for selenium or selenoproteins in regulating EGF and insulin receptor tyrosine kinase activity and suggest a mechanism whereby selenium‐containing compounds contribute to cell growth.
X‐ray crystal structure of the serine proteinase inhibitor eglin c at 1.95 Å resolution
Authors: Hipler Karsten , Priestle John P. , Rahuel Joseph , Grütter Markus G.
Published: July 2002

The crystal structure eglin c. naturally occurring in the leech Hirudo medicinalis, is known from its complexes with various serine proteinases, but the crystallization of free eglin c has not yet been reported. A method is described for growing well‐diffracing crystals of free eglic c from highly concentrated protein solutions (≈2OO mg/ml). The space group of the orthorhombic crystals was determined to be P212121 with unit cell parameters a = 32.6. b = 42.0. c = 44.1 Å. The structure or free eglin c was resolved at 1.95 Å resolution by Patterson search methods. The final model contains all 70 amino acids of eglin c and 125 water molecules. In comparison to the eglin structure known from its complexes with proteinases, only small differences have been observed in free eglin c. However, the reactive site‐binding loop and a few residues on the surface of eglin have been found in different conformations due to crystal contacts. In contrast to the complex structures, the first seven amino acids of the highly flexible amino terminus can be located. Crystallographic refinement comprised molecular dynamics refinement, classical retrained least‐squares refinement and individual isotropic atomic temperature refinement. The final R‐factor is 15.8%.
Modulation of the glucagon‐dependent activation of the phosphoenolpyruvate carboxykinase gene by oxygen in rat hepatocyte cultures Evidence for a heme protein as oxygen sensor
Authors: Kietzmann Thomas , Schmidt Helga , Probst Irmelin , Jungermann Kurt
Published: July 2002

The glucagon‐dependent activation of the phosphoenolpyruvate carboxykinase (PCK) gene is modulated by oxygen. It was proposed that heme proteins might function as O2 sensors; their actions are impaired after replacement of the central Fe2+ ion by Co2+ and inhibition of heme synthesis by succinylacetone (SA). Therefore, the effects of CoCl2 and SA, alone and in combination, on the glucagon‐dependent induction of PCK activity and PCK mRNA were investigated at different physiological oxygen tensions in primary rat hepatocyte cultures. The cells were exposed to 50 μM CoCl2 and/or 2 mM SA from 4–24 h. After addition of fresh media without CoCl2 or SA, PCK was induced with 1 nM glucagon, PCK activity and PCK mRNA were elevated to 100% at 16% O2 and to about 63% at 8% O2, CoCl2 reduced these increases to about 45% at 16% O2 and to about 35% at 8% O2. SA lowered the inductions to about 50% and 40% each at 16% and 8% O2. CoCl2 plus SA diminished the elevations to about 5% at both oxygen tensions. In the presence of CoCl2 and/or SA, ornithine decarboxylase induction by insulin was not impaired; lactate dehydrogenase did not leak from the cells, which in electron microscopical inspections had normal cell structures. These findings support the hypothesis that a heme protein is involved in the activation of the PCK gene and that it acts as an O2 sensor.
Localization of the steroid‐binding site of the human sex steroid‐binding protein of plasma (SBP or SHBG) by site‐directed mutagenesis
Authors: Sui Li-ming , Cheung Audrey W.C. , Namkung Pearl C. , Petra Philip H.
Published: July 2002

The amino‐terminal region of the human sex steroid‐binding protein of plasma (SBP or SHBG) containing K134 and M139 was found to represent part of the steroid‐binding site. This was accomplished by constructing and expressing site‐directed mutants having the following replacements: M139L, M139K, M139S, K134A, H235S, and Y57F. The results indicated that M139L and H235S were fully‐active, K134A and Y57F were 50 and 67% active, M139K was 7% active, and M 139S was inactive. These results support affinity‐labeling data indicating that both K134 and M139 are located in or near the site, and suggest that Y57 may play a role in steroid binding. The fully active H235S mutant reveals that H235 is not involved in the steroid‐binding process.
A subgroup of class α glutathione S‐transferases Cloning of cDNA for mouse lung glutathione S‐transferase GST 5.7
Authors: Zimniak Piotr , Eckles Michael A. , Saxena Manju , Awasthi Yogesh C.
Published: July 2002

A full‐length cDNA clone encoding the previously purified mouse glutathione S‐transferase GST 5.7 [(1991), Biochem. J. 278, 793–799] has been isolated from a mouse lung cDNA library in λgt11. Sequencing of the clone revealed the presence of microheterogeneity in GST 5.7. Comparison of the deduced protein sequence with other glutathione S‐transferases, together with previous information available on GST 5.7, indicates that the enzyme belongs to a novel subgroup within the α class of glutathione S‐transferases. Members of the subgroup, which also include the rat GST 8‐8 and perhaps chicken GST CL3, show high sequence homology with each other, but only moderate similarity to other α class enzymes. They share a substrate specificity profile that resembles π‐class enzymes, and are active in the conjugation of lipid peroxidation products.
Transcriptional induction of the fatty acid binding protein gene in mouse liver by bezafibrate
Authors: Besnard P. , Mallordy A. , Carlier H.
Published: July 2002

The mechanism by which hypolipidemic peroxisome proliferators of the fibrate family induce the liver fatty acid binding protein in liver of rodents is unknown. In order to delineate the level at which this protein is induced, the transcriptional activity of the specific gene encoding for liver fatty acid binding protein was measured in isolated hepatocyte nuclei obtained from male Swiss mice daily force‐fed during 7 days with 400 mg/kg body weight bezafibrate. This treatment induced a 4‐fold increase in the liver fatty acid binding protein transcription rate. Liver fatty acid binding protein mRNA level, measured by Northern blot analysis and cytosolic content of this protein, analyzed by immunoblotting, increased concurrently. From these results we conclude that the increase in the cytosolic liver fatty acid binding protein level by bezafibrate is due to an enhancement of the transcription rate of the liver fatty acid binding protein gene. Whether the transcriptional effect is mediated by peroxisome proliferator‐receptor remains to be elucidated.
Growth hormone mediates zone‐specific gene expression in liver
Authors: Oinonen T. , Nikkola E. , Lindros K.O.
Published: July 2002

The CYP2B1/2 gene pair is an example of many liver genes that exhibit a characteristic zonated expression pattern in the liver. The factors governing this zonation are poorly understood. We observed that after hypophysectomy the expression of CYP2B1/2 protein and mRNA in the normally silent periportal (upstream) region was high, in both male and female rats. Treatment with growth hormone counteracted the effect of hypophysectomy, limiting expression to the perivenous (downstream) region, completely in females and partially in males. This shows that a hormone zone‐specifically regulates gene expression in the liver.
Cloning of a human liver UDP‐glucose pyrophosphorylase cDNA by complementation of the bacterial galU mutation
Authors: Peng Hwei-Ling , Chang Hwan-You
Published: July 2002

A human liver cDNA clone which encodes the UDP‐glucose pyrophosphorylase was isolated by complementation of a bacterial galU mutant. The deduced amino acid sequence of the human enzyme comprised 508 amino acids with a calculated molecular mass of 56,950. The human enzyme significantly resembles those of potato tuber and slime mold with a homology of 46.6% and 43.2%, respectively, in amino acid sequence. No homology was found between the eukaryotic and the prokaryotic enzymes. Northern blotting analysis revealed that the gene was expressed at the highest level in skeletal muscle, followed by liver, heart and kidney.
Elongation in a Dictyostelium in vitro translation system is affected by calmodulin antagonists
Authors: Sonnemann Jürgen , Drugeon Gabrielle , Haenni Anne-Lise , Mutzel Rupert
Published: July 2002

We have previously shown that the Dictyostelium discoideum ribosomal protein L19 specifically binds Ca2+/calmodulin [Sonneman et al. (1991) J. Biol. Chem. 266, 23091‐23096]. To investigate the role of calmodulin in the regulation of protein synthesis, we have now established an in vitro protein synthesizing system from Dictyostelium cells which can elongate polypeptide chains with high efficiency. Various calmodulin antagonists affected translation in this system. The inhibitory effects of the antagonists could be partially reversed by addition of calmodulin. A monoclonal antibody against D. discoideum calmodulin also specifically inhibited protein synthesis. Similar effects of calmodulin antagonists were found in a standard wheat germ in vitro translation system.
Time‐resolved study of tryptophan fluorescence in vesicle reconstituted cytochrome oxidase
Authors: Das Tapan K. , Mazumdar Shyamalava
Published: July 2002

Time‐resolved study of fluorescence decay of the tryptophan residue in bovine cytochrome c oxidase in phospholipid vesicles is reported for the first time. The effect of the redox state of the protein on its conformation has been investigated using time‐resolved decay of tryptophan fluorescence in the oxidised and reduced protein. The fluorescence decay was best fitted using a discrete three exponential model. Amplitude distribution of lifetimes also showed three distinct regions in the analysis of decay profiles by the maximum entropy method (MEM). Results of the time resolved studies showed that the amplitudes as well as the lifetimes of the trytophan fluorescence remain the same for the oxidised and the reduced states ofcytoehrome c oxidase, indicating that the environment around tryptophan residues remains more or less unaltered on reduction of the protein. The results suggest that there is no global conformational change in the protein on electron transfer and support the possibility of the existence of local fluctuations in the protein during the redox cycle. p]Cytochrome c oxidase; Tryptophan fluorescence; Conformational change
Structural study of rat thyroid transcription factor 1 homeodomain (TTF‐1 HD) by nuclear magnetic resonance
Authors: Viglino Paolo , Fogolari Federico , Formisano Silvestro , Bortolotti Nadia , Damante Giuseppe , Di Lauro Roberto , Esposito Gennaro
Published: July 2002

The 500 MHz 1H NMR spectrum of a 68‐residue peptide, encompassing the rat thyroid transcription factor 1 homeodomain (TTF‐1 HD), was fully assigned using standard 2D NMR methodology. The secondary structure elements and their spatial organization were determined and led to a structure very similar to that previously described for other homeodomains and expected also for TTF‐1 HD from homology modeling predictions. The three‐dimensional arrangement of the three helix fragments of TTF‐1 HD preserves the helix‐tum‐helix motif commonly occurring in many classes of DNA‐binding proteins.
Synchronous replication of SV 40 DNA in virus infected TC 7 cells induced by transient hypoxia
Authors: Dreier Torsten , Scheidtmann Karl Heinz , Probst Hans
Published: July 2002

We transiently exposed SV 40 infected TC 7 cell cultures to a reduced O2 tension (4–8 h, about 200 ppm relative to 105 Pa total pressure). Under the hypoxic conditions, ‘working’ viral replication forks were greatly retarded or stopped, and initiation of daughter strand synthesis in further SV 40 DNA molecules was suppressed. Reoxygenation released an immediate burst of SV 40 replication which mainly consisted of a synchronous viral replication round. This synchronous in vivo replication began at the known origin of replication and proceeded at normal rates to the known termination region. Viral replicons seemed to accumulate under hypoxia in a state fully prepared to begin replication immediately after recovery of a normal pO2. The shut‐down and sudden reactivation of DNA synthesis under hypoxia and reoxygenation, respectively, were not accompanied by changes of the phosphorylation state of large T antigen. The described synchronization procedure can be applied to optionally large SV 40 infected cell cultures.
Classification of fumarate reductases and succinate dehydrogenases based upon their contrasting behaviour in the reduced benzylviologen/fumarate assay
Authors: Ackrell Brian A.C. , Armstrong Fraser A. , Cochran Bruce , Sucheta Artur , Yu Tao
Published: July 2002

Reduction of fumarate by soluble beef heart succinate dehydrogenase has been shown previously by voltammetry to become increasingly retarded as the potential is lowered below a threshold potential of −80 mV at pH 7.5. The behaviour resembles that of a tunnel diode, an electronic device exhibiting the property of negative resistance. The enzyme thus acts to oppose fumarate reduction under conditions of high thermodynamic driving force. We now provide independent evidence for this phenomenon from spectrophotometric kinetic assays. With reduced benzylviologen as electron donor, we have studied the reduction of fumarate catalysed by various enzymes classified either as succinate dehydrogenases or fumarate reductases. For succinate dehydrogenases, the rate increases as the concentration of reduced dye (driving force) decreases during the reaction. In contrast, authentic fumarate reductases of anaerobic cells (and ‘succinate dehydrogenase’ from Bacillus subtilis) neither exhibit the electrochemical effect nor deviate from simple kinetic behaviour in the cuvette assay. The ‘tunnel‐diode’ effect may thus represent an evolutionary adaptation to aerobic metabolism.
Crystal structure of transketolase in complex with thiamine thiazolone diphosphate, an analogue of the reaction intermediate, at 2.3 Å resolution
Authors: Nilsson Ulrika , Lindqvist Ylva , Kluger Ronald , Schneider Gunter
Published: July 2002

The crystal structure of the complex of transketolase and thiamine thiazolone diphosphate has been determined at 2.3 Å resolution. The complex has a structure which closely resembles that of this enzyme with the cofactor ThDP. This is consistent with the observation that the binding of the analogue to transketolase involves ground state rather than transition state interactions. Since thiamine thiazolone diphosphate resembles an expected intermediate in the catalytic pathway, the structure of the intermediate was modelled from the crystal structure. Based on this model, enzymic groups responsible for binding of the intermediate and proton transfer during catalysis are suggested.
Structure, transmembrane topology and helix packing of P‐type ion pumps
Authors: Stokes David L. , Taylor William R. , Green N.Michael
Published: July 2002

Electron microscopy has recently provided improved structures for P‐type ion pumps. In the case of Ca2+‐ATPase, the use of unstained specimens revealed the structure of the transmembrane domain. The composition of this domain has been controversial due to the variety of methods used to study the number and exact locations of transmembrane crossings within the sequence. After reviewing the results from several members of the family, we found a consensus for 10 transmembrane segments, and also that 10 helices fitted well into the structure of Ca2+‐ATPase. Thus, we present the most detailed model for transmembrane structure so far, in the hope of stimulating more precise experimental strategies.
Purification and spectroscopic characterization of a recombinant amino‐terminal polypeptide fragment of mouse epithelial cadherin
Authors: I. Tong Kit , Yau Patrick , Overduin Michael , Bagby Stefan , Porumb Tudor , Takeichi Masatoshi , Ikura Mitsuhiko
Published: July 2002

Cadherins are a family of Ca2+‐dependent cell adhesion molecules containing four extracellular tandem repeats each of 110 amino acids. The most amino‐terminal repeat is believed to confer the specificity of cell adhesion. A polypeptide containing the amino‐terminal repeat of mouse epithelial cadherin has been over‐expressed in E. coli and purified to homogeneity. This polypeptide binds Ca2+ with a dissociation constant of 1.6 × 10−4M. CD and NMR experiments indicate that the polypeptide adopts a predominantly β‐sheet conformation and that binding of Ca2+ induces only small conformational changes.
Effect of unmodified triple helix‐forming oligodeoxyribonucleotide targeted to human multidrug‐resistance gene mdr1 in MDR cancer cells
Authors: Scaggiante Bruna , Morassutti Carla , Tolazzi Giuseppe , Michelutti Angela , Baccarani Michele , Quadrifoglio Franco
Published: July 2002

The human mdr1 gene encodes a transmembrane glycoprotein the over‐expression of which is associated with development of multidrug resistance in human tumor cells. A negative modulation of human mdr1 has been attempted via a 27‐mer unmodified triple helix‐forming oligonucleotide, named 1D, targeted to a homopurine sequence in the coding region of the gene. By administering 10 μM of 1D we could find a significant reduction in MDR1 mRNA levels in the human drug‐resistant cell line CEM‐VLB 100. This effect appears to be specific and due to a transient block of RNA polymerase mediated by triple helix formation.
pH‐independent inhibition of restriction endonuclease cleavage via triple helix formation by oligonucleotides containing 8‐oxo‐2′‐deoxyadenosine
Authors: Wang Qing , Tsukahara Satoru , Yamakawa Hidefumi , Takai Kazuyuki , Takaku Hiroshi
Published: July 2002

The ability of homopyrimidine oligonucleotides containing 8‐oxo‐2′‐deoxyadenosine to form stable, triple helical structures with the sequence containing the recognition site for the class II‐S restriction enzyme, Ksp632‐I, was studied as a function of pH. The 8‐oxo‐2′‐deoxyadenosine‐substituted oligomers were shown to inhibit enzymatic cleavage and to bind within the physiological pH range in a pH‐independent fashion without compromising specificity.
The reversion of highly tumorigenic cell lines to non‐tumorigenic phenotype is associated with c‐jun down‐expression
Authors: Lavrovsky Yan , Yefremov Yaroslav , Lavrovsky Vadim
Published: July 2002

Using model spontaneously reverting cell lines, c‐jun, junB, junD and c‐fos oncogene expression was investigated. c‐jun, but not junB, junD or c‐fos, was overexpressed in highly tumorigenic clones. The reversion of cells to the non‐tumorigenic phenotype resulted in a dramatic decrease in c‐jun expression. CAT assays revealed that c‐jun overexpression in tumorigenic cells was associated with higher transcription activity. No correlation between c‐jun oncogene expression and AP‐1 transcription factor activity in tumorigenic and non‐tumorigenic clones was found.
Repression of in vitro transcription of the Escherichia coli fnr and nar X genes by FNR protein
Authors: Takahashi Kojiro , Hattori Takako , Nakanishi Tohru , Nohno Tsutomu , Fujita Nobuyuki , Ishihama Akira , Taniguchi Shigehiko
Published: July 2002

In facultative anaerobes, the anaerobic expression of respiratory genes is regulated by a transcriptional activator, FNR. Transcription in vitro of the E. coli fnr gene was repressed by its product, FNR. The transcription of the E. coli nar X gene encoding the nitrate sensor protein was likewise repressed. DNA truncation experiments for fnr and nar X genes indicated that multiple anaero‐boxes in each promoter region are essential for repression by the FNR protein, but they also suggest that factor‐independent upstream activation signals are operating with these promoters.
A human B‐box‐binding protein downregulated in adenovirus 5‐transformed human cells
Authors: Kropotov A.V. , Tomilin N.V.
Published: July 2002

Internal promoters of some genes transcribed by RNA polymerase III (e.g. tRNA genes, adenovirus VA1 RNA gene, human retroposons of the Alu family) contain a conserved sequence element, B‐box, interacting with basal transcription factor TFIIIC2 which initiates assembly of the full transcription complex on the genes and which represents the major determinant of the efficiency of their expression. In this study we have identified in human nuclear extracts a protein which interacts with VA1 B‐box DNA and forms a high‐affinity complex which is very stable after the addition of a large excess of competitor DNA. Unlike TFIIIC2, the B‐box‐binding activity of the B‐box‐binding protein is found to be decreased in adenovirus 5‐transformed human cells. In these cells (line 293) increased transcription of VA1 and tRNA genes in vivo and in vitro was previously detected by other workers. Our results suggest that besides TFIIIC2, an additional B‐box‐binding protein factor may be involved in the regulation of expression of the RNA polymerase III‐transcribed genes.
Characterisation, crystallisation and preliminary X‐ray diffraction analysis of a Fab fragment of a rat monoclonal antibody with very high affinity for the human muscle acetylcholine receptor
Authors: Kontou Maria , Vatzaki Efstratia H. , Kokla Anna , Acharya K.Ravi , Oikonomakos Nikos G. , Tzartos Socrates J.
Published: July 2002

The Fab fragment of a rat monoclonal antibody (no. 192) with very high affinity for the main immunogenic region of the human muscle nicotinic acetylcholine receptor (AChR) has been purified, characterised and crystallised using vapour diffusion techniques. Its Kd for human AChR was determined to be 5×10−11 M. Its cross‐reactivity pattern suggests that residue α23 of the AChR strongly affects its epitope. Crystals suitable for X‐ray analysis, obtained by micro‐ and macroseeding techniques, belong to the orthorhombic space group C2221 and they diffract to 2.8 A resolution using synchrotron radiation. The unit cell dimensions are α = 83.4 Å, and and there are two Fab molecules per asymmetric unit.
Contrast sonography for inconclusive findings on routine sonography
Authors: Gücer F. , Häusler M.C.H. , Arikan M.G. , Pieber D.
Published: July 2002

Objectives: We conducted a pilot study in 20 women with sonographically suspect endometria, to assess the value of contrast sonography and patient acceptance of this procedure. Methods: Saline solution 4–20 ml was injected into the uterine cavity using an embryo transfer catheter, followed by hysteroscopy in 19 cases and hysterectomy in one case. Results: A polyp was diagnosed in 12 patients, a submucous myoma in one patient, a proliferated endometrium in five patients and a placental polyp in one patient. A sonographic irregular structure was diagnosed in one patient which turned out to be coagula on hysteroscopy and histology. The procedure was well accepted by all patients. The diagnosis found by contrast sonography agreed in all cases with that found by hysteroscopy. Conclusion: Our results show that contrast sonography is an easy, quick and inexpensive procedure which increases the diagnostic value of vaginal sonography. The indications for contrast sonography are based on inconclusive sonographic findings, especially if polyps or submucous myoma are suspected.
A viable ubiquitin‐activating enzyme mutant for evaluating ubiquitin system function in Saccharomyces cerevisiae
Authors: Swanson Robert , Hochstrasser Mark
Published: July 2002

Ligation of proteins to ubiquitin requires activation of ubiquitin by E1, the ubiquitin‐activating enzyme. Mutant alleles of E1 in mammalian cells have been crucial for dissecting the contribution of the ubiquitin system to cell function. Comparable mutants have been unavailable for Saccharomyces cerevisiae. Here we describe the isolation and characterization of a hypomorphic allele of S. cerevisiae E1. Protein modification by ubiquitin is strongly impaired in the mutant, inhibiting degradation of ubiquitin–proteasome pathway substrates as well as ubiquitin‐dependent but proteasome‐independent degradation of membrane receptors. This allele will be a useful tool for evaluating the ubiquitin‐dependence of cellular processes in yeast, even those in which the proteasome is not involved.
Heterogeneous halothane binding in the SR Ca2+‐ATPase
Authors: Kosk-Kosicka Danuta , Fomitcheva Ioulia , Lopez Maria M , Eckenhoff Roderic G
Published: July 2002

The activity of various Ca2+‐ATPases is affected by volatile anesthetics, such as halothane, commonly used in clinical practice. The effect on the enzyme in skeletal muscle sarcoplasmic reticulum (SR) is biphasic, including stimulation at clinical anesthetic concentrations and subsequent inhibition at higher concentrations. We have previously proposed that the action of a volatile anesthetic on Ca2+‐ATPases results from its binding in the interior of the enzyme molecule [Lopez, M.M. and Kosk‐Kosicka, D. (1995) J. Biol. Chem. 270, 28239–28245]. Presently, we investigated whether the anesthetic interacts directly with the skeletal muscle SR Ca2+‐ATPase (SERCA1) as evidenced by binding. Photoaffinity labeling with [14C]halothane demonstrated that the anesthetic binds saturably to SR membranes, and that ≈80% of the binding is specific, with a K
I of 0.6 mM. The K
I value agrees well with the concentration at which halothane half‐maximally activates SERCA1. SDS gel electrophoresis of labeled membranes indicates that 38–56% of [14C]halothane incorporates into SERCA1, and 38–53% in lipids. Distribution of label among the three fragments produced by controlled tryptic digestion of SERCA1 suggests heterogeneous halothane binding presumably in discrete sites in the enzyme. The results provide the first direct evidence that halothane binds to SERCA1. Potentially this binding could be related to anesthetic effect on enzyme's function.
Highly purified D1/D2/cyt b559 preparations from spinach do not contain the non‐heme iron center
Authors: Kurreck J. , Garbers A. , Parak F. , Renger G.
Published: July 2002

© 1997 Federation of European Biochemical Societies.
The structure of porin from Paracoccus denitrificans at 3.1 Å resolution
Authors: Hirsch A. , Breed J. , Saxena K. , Richter O-M.H. , Ludwig B. , Diederichs K. , Welte W.
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Substrate inhibition of cruzipain is not affected by the C‐terminal domain
Authors: Stoka Veronika , McKerrow James H , Cazzulo Juan José , Turk Vito
Published: July 2002

Endogenous and recombinant cruzipain, the major cysteine proteinase from the protozoan parasite Trypanosoma cruzi, exhibit differences in the protein and circular dichroism spectra probably attributed to the absence of the C‐terminal domain in the recombinant enzyme. Substrate hydrolysis of both molecules at 25°C and neutral pH obeyed Michaelis‐Menten kinetics whereas significant substrate inhibition was observed above neutral pH. The results suggest that substrate inhibition of cruzipain is pH‐dependent, and that the C‐terminal domain does not play an essential role in this process.
Retinoic acid regulates selectively the expression of immediate early response genes in PC12 cells
Authors: Cosgaya José Miguel , Pérez-Juste German , Aranda Ana
Published: July 2002

Nerve growth factor (NGF) induces neuronal differentiation and growth arrest in PC12 cells. One of the initial effects of NGF in these cells is the induction of the expression of immediate early genes (IEGs). In this study we have analyzed the influence of retinoic acid (RA), which exerts important effects on PC12 cell proliferation and function, on the expression of IEGs. Incubation with RA did not alter NGFI‐A mRNA levels, but significantly reduced the NGFI‐B and c‐fos response to NGF and serum. The response to NGF was maximal in the presence of cycloheximide, and RA also reduced the superinduction of NGFI‐B and c‐fos mRNA levels. Sequences contained within the 5′ flanking region of the c‐fos gene confer responsiveness to NGF and mediate the inhibitory effect of RA. The differential regulation by RA suggests that NGF induces expression of the three IEGs by different mechanisms.
Differences in transition state stabilization between thermolysin (EC 3.4.24.27) and neprilysin (EC 3.4.24.11)
Authors: Marie-Claire Cynthia , Ruffet Emmanuel , Tiraboschi Gilles , Fournie-Zaluski Marie-Claude
Published: July 2002

Important homologies in the topology of the catalytic site and the mechanism of action of thermolysin and neprilysin have been evidenced by site‐directed mutagenesis. The determination of differences in transition state stabilization between these peptidases could facilitate the design of specific inhibitors. Thus, two residues of thermolysin which could be directly (Tyr157) or indirectly (Asp226) involved in the stabilization of the transition state and their putative counterparts in neprilysin (Tyr625 and Asp709) have been mutated. The results show that Tyr157 is important for thermolysin activity while Tyr625 has no functional role in neprilysin. Conversely, the mutation of Asp226 induced a slight perturbation of thermolysin activity while Asp709 in neprilysin seems crucial in neprilysin catalysis. Taken together these data seem to indicate differences in the transition state mode of stabilization in the two peptidases.
Different phosphate binding modes of Streptomyces griseus aminopeptidase between crystal and solution states and the status of zinc‐bound water
Authors: Harris Michael N , Ming Li-June
Published: July 2002

Phosphate shows a non‐competitive inhibition toward a Streptomyces aminopeptidase (sAP) between pH 5.85 (K
i=0.48 mM) and 9.0 (110 mM), with a pK
a of 7.1 likely due to ionization of H2PO4
−. This non‐competitive inhibition pattern indicates that phosphate binding to sAP in solution is different from that in the crystal structure, where phosphate is bound to the active site Zn(II) ions. Fluoride uncompetitively inhibits sAP from pH 5.5 (K
i=3.72 mM) to 9.0 (43.6 mM), with a pK
a of ∼6.2 likely due to a coordinated water. The different inhibition natures and pK
a values indicate that the two inhibitors bind at different locations.
High level expression of Thermococcus litoralis 4‐α‐glucanotransferase in a soluble form in Escherichia coli with a novel expression system involving minor arginine tRNAs and GroELS
Authors: Imamura Hiromi , Jeon Beong-Sam , Wakagi Takayoshi , Matsuzawa Hiroshi
Published: July 2002

The Thermococcus litoralis 4‐α‐glucanotransferase (GTase) gene has a high content of AGA and AGG codons for arginine, which are extremely rare in Escherichia coli. Expression of the GTase gene in E. coli resulted in low protein production and the accumulation of inclusion bodies. However, simultaneous expression of GTase with tRNAAGA, tRNAAGG and GroELS affected both the production and solubility of GTase, and production of soluble GTase increasing about 5‐fold. This new E. coli expression system should be applicable to the expression of not only archaeal but also eukaryotic genes, which usually contain a large number of AGA and AGG codons.
Metabolite transport in isolated yeast mitochondria: fumarate/malate and succinate/malate antiports
Authors: Pallotta Maria Luigia , Fratianni Alessandra , Passarella Salvatore
Published: July 2002

In this study, we investigated the metabolite permeability of isolated coupled Saccharomyces cerevisiae mitochondria. The occurrence of a fumarate/malate antiporter activity was shown. The activity differs from that of the dicarboxylate carrier (which catalyses the succinate/malate antiport) in (a) kinetics (K
m and V
max values are about 27 μM and 22 nmol min−1 mg protein−1 and 70 μM and 4 nmol min−1 mg protein−1, respectively), (b) sensitivity to inhibitors, (c) K
i for the competitive inhibitor phenylsuccinate and (d) pH profiles.
Metal regulation of metallothionein participation in redox reactions
Authors: Zhang Sen , Li Jian , Wang Chih-Chen , Tsou Chen-Lu
Published: July 2002

Like glutathione or dithiothreitol, metallothionein effects the formation of pancreatic ribonuclease A from its S‐sulfonated derivative catalyzed by protein disulfide isomerase. EDTA increases the yield of ribonuclease A activity recovery with metallothionein but does not affect the reaction with glutathione or dithiothreitol. EDTA also increases the reactivity of thiol groups in metallothionein with 5,5′‐dithiobis‐(2‐nitrobenzoic acid) by chelation of zinc ions. It is suggested that the thiol groups in metallothionein form a part of the pool of cellular thiols in the regulation of cellular redox reactions and their availability is modulated by zinc chelation.