bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic transfer of the pigment bacteriorhodopsin into the eukaryote Schizosaccharomyces pombe
Authors: Hildebrandt Volker , Ramezani-Rad Massoud , Swida Ulrike , Wrede Paul , Grzesiek Stephan , Primke Marion , Büldt Georg
Published: July 2002

The gene encoding for bacterio‐opsin (bop gene) from Halobacterium halobium has been introduced in a yeast expression vector. After transformation in Schizosaccharomyces pombe, bacterio‐opsin (BO) is expressed and was detected by antisera. The precursor protein of BO (pre‐BO) is processed by cleavage of aniino acids at the N‐terminal end as in H. halobium. Addition of the chromophore, retinal, to the culture medium results in a slight purple colour of the yeast cells indicating the in vivo regeneration of BO to bacteriorhodopsin (BR) and its incorporation into membranes. Therefore, in contrast to the expression in E. coli, isolation of the membrane protein and reconstitution in lipid vesicles is not necessary for functional analysis. The kinetics of the ground state signal of the photocycle BR in protoplasts is demonstrated by flash spectroscopy and is comparable to that of the natural system. The present investigation shows for the first time the transfer of an energy converting protein from archaebacteria to eukaryotes by genetic techniques. This is a basis for further studies on membrane biogenesis, genetics, and bioenergetics by analysis of in vivo active mutants.
The influence of stereospecific assignments on the determination of three‐dimensional structures of proteins by nuclear magnetic resonance spectroscopy
Authors: Driscoll Paul C. , Gronenborn Angela M. , Clore G.Marius
Published: July 2002

The influence of the stereospecific assignments of β‐methylene protons and the classification of χ1 torsion angles on the definition of the three‐dimensional structures of proteins determined from NMR data is investigated using the sea anemone protein BDS‐I (43 residues) as a model system. Two sets of structures are computed. The first set comprises 42 converged structures (denoted STEREO structures) calculated on the basis of the complete list of restraints derived from the NMR data, consisting of 489 interproton and 24 hydrogen bonding distance restraints, supplemented by 23 ϕ backbone and 21 χ1 side chain torsion angle restraints. The second set comprises 31 converged structures (denoted NOSTEREO structures) calculated from a reduced data set in which those restraints arising from stereospecific assignments, and the corresponding χ1 torsion angle restraints, are explicitly omitted. The results show that the inclusion of the stereospecific restraints leads to a significant improvement in the definition of the structure of BDS‐I, both with respect to the backbone and the detailed arrangement of the side chains. Average atomic rms differences between the individual structures and the mean structures for the backbone atoms are 0.67±0.12 Å and 0.93±0.16 Å for the STEREO and NOSTEREO structures, respectively; the corresponding values for all atoms are 0.90±0.17 Å and 1.17±0.17 Å, respectively. In addition, while the overall fold remains unchanged, there is a small but significant atomic displacement between the two sets of structures.
Identification of the magnesium, europium and lead binding sites in E. coli and lupine tRNAPhe by specific metal ion‐induced cleavages
Authors: Marciniec Tadeusz , Ciesiołka Jerzy , Wrzesinski Jan , Krzyżosiak Włodzimierz J.
Published: July 2002

The Pb, Eu and Mg‐induced cleavages in E. coli and lupine tRNAPhe have been characterized and compared with those found in yeast tRNAPhe. The pattern of lupine tRNAPhe hydrolysis closely resembles that of yeast tRNAPhe, while several major differences occur in the specificity and efficiency of the E. coli tRNAPhe hydrolysis. The latter tRNA is cleaved with much lower yield in the D‐loop, and interestingly, cleavage is also detected in the variable region, that is highly resistant to hydrolysis in eukaryotic tRNAs. The possible location of tight Pb, Eu and Mg binding sites in E. coli tRNAPhe is discussed on the basis of the specific hydrolysis data.
Isolated fungal cellulose terminal domains and a synthetic minimum analogue bind to cellulose
Authors: Johansson Gunnar , Ståhlberg Jerry , Lindeberg Gunnar , Engström Åke , Pettersson Göran
Published: July 2002

The cellulose‐binding properties of the highly conserved terminal region which is common to several fungal cellulases were studied. Domains were prepared by proteolytic cleavage of Trichoderma reesei CBH1 and the corresponding enzyme from Sporotrichum pulverulentum, and a peptide corresponding to residues 462–497 (the C‐terminal part) of Trichoderma CBH1 was synthesized. The three peptides showed similar binding behavior, whereas reduced and S‐carboxymethylated T. reesei fragment was inactive. This region thus appears to serve as an independent functional domain in which the C‐terminal part is responsible for the binding, which in turn requires an intact three‐dimensional structure.
Low molecular mass GTP‐binding proteins of adrenal chromaffin cells are present on the secretory granule
Authors: Burgoyne Robert D. , Morgan Alan
Published: July 2002

Adrenal medullary homogenates and chromaffin granule membranes were separated by SDS‐polyacrylamide gel electrophoresis and GTP‐binding proteins detected using [α‐32P]GTP binding to nitrocellulose blots. Four GTP‐binding polypeptides of 24, 22, 20 and 18 kDa were routinely found in medullary homogenates and all were also found in isolated chromaffin granule membranes. The GTP‐binding polypeptides co‐sedimented with granule membrane markers following separation on sucrose gradients. On the basis of trypsin sensitivity and resistance to extraction, the GTP‐binding proteins appeared to be tightly bound to the cytoplasmic surface of the granules. One or more of the secretory granule GTP‐binding proteins could be involved in exocytosis in adrenal chromaffin cells.
On the biosynthesis of free and covalently bound PQQ Glutamic acid decarboxylase from Escherichia coli is a pyridoxo‐quinoprotein
Authors: van der Meer Robert A. , Groen Barend W. , Duine Johannis A.
Published: July 2002

Analysis of glutamic acid decarboxylase (GDC) (EC 4.1.1.15) from Escherichia coli ATCC 11246 revealed the presence of six pyridoxal phosphates (PLPs) as well as six covalently bound pyrroloquinoline quinones (PQQs) per hexameric enzyme molecule. This is the second example of a pyridoxo‐quinoprotein, suggesting that other atypical pyridoxoproteins (PLP‐containing enzymes) have similar cofactor composition. Since the organism did not produce free PQQ and its quinoprotein glucose dehydrogenase was present in the apo form, free PQQ is not used in the assemblage of GDC. Most probably, biosynthesis of covalently bound cofactor occurs in situ via a route which is different from that of free PQQ. Thus, organisms previously believed to be unable to synthesize (free) PQQ could in fact be able to produce quinoproteins with covalently bound cofactor. Implications for the role of PQQ in eukaryotic cells are discussed.
Protein synthesis inhibitors and catalytic RNA Effect of puromycin on tRNA precursor processing by the RNA component of Escherichia coli RNase P
Authors: Vioque Agustin
Published: July 2002

RNase P and ribosomes must interact with similar substrate molecules, tRNA precursors in the case of RNase P and aminoacyl‐, peptidyl‐ or free tRNAs in the case of ribosomes. In order to compare the substrate recognition mechanisms between ribosomes and RNase P, protein synthesis inhibitors have been assayed for their effect on the catalytic activity of the RNA component of Escherichia coli RNase P (M1 RNA). Puromycin has an inhibitory effect that could be related to similar substrate recognition mechanisms by rRNA in the ribosome and by M1 RNA in RNase P.
Nonmuscle actin ADP‐ribosylated by botulinum C2 toxin caps actin filaments
Authors: Weigt Christiane , Just Ingo , Wegner Albrecht , Aktories Klaus
Published: July 2002

The effect of nonmuscle actin ADP‐ribosylated by botulinum C2 toxin on the polymerization of nonmuscle actin was investigated in order to clarify whether nonmuscle actin is converted into a capping protein by ADP‐ribosylation. ADP‐ribosylated actin was found to decrease the rate of polymerization of actin filaments which are free at both ends. ADP‐ribosylated actin turned out to have no effect on the rate or extent of polymerization at the pointed ends of actin filaments the barbed ends of which were capped by gelsolin. The monomer concentration reached at the final stage of polymerization was similar to the critical concentration of the pointed ends of actin filaments. The results suggest that nonmuscle actin ADP‐ribosylated by botulinum C2 toxin acts as a capping protein which binds to the barbed ends to inhibit polymerization.
Calcium regulates the rate of rhodopsin disactivation and the primary amplification step in visual transduction
Authors: Wagner Rafael , Ryba Nicholas , Uhl Rainer
Published: July 2002

The kinetics of the light‐induced activation of transducin as well as the subsequent disactivation process can be monitored by means of a specific light scattering transient PA. In this communication it is demonstrated that the rate of transducin disactivation is calcium dependent, increasing when the calcium concentration is decreased. As a consequence of the accelerated recovery in low calcium, the time to the peak of the transducin activation process is shortened and the gain of the primary amplification step, i.e. the number of transducin molecules activated per bleached rhodopsin, is reduced. Experiments using hydroxylamine as an artificial quencher of rhodopsin activity suggest that calcium acts upon rhodopsin kinase and not upon the rate of the GTPase. This would indicate that calcium may control visual adaptation not only by regulating guanine cyclase activity, but also by affecting the primary step in the transduction cascade, the rhodopsin‐transducin coupling
Crystallization and preliminary X‐ray analysis of porin from Rhodobacter capsulatus
Authors: Nestel Uwe , Wacker Thomas , Woitzik Daniela , Weckesser Jürgen , Kreutz Werner , Welte Wolfram
Published: July 2002

Porin monomers of the phototrophic bacterium Rhodobacter capsulatus were purified. Crystals were obtained from a solution of porin solubilized with the detergent octyltetraoxyethylene within 5 days using the vapor phase equilibration technique. The crystals were rhombohedral with an edge length of 0.4 mm. The space group was trigonal R3 with unit cell constants of a=b=95 Å, c=147 Å. Reflexions were observed to 3.2 Å.
Evidence for a PO2‐sensitive K+ channel in the type‐I cell of the rabbit carotid body
Authors: Delpiano M.A. , Hescheler J.
Published: July 2002

Type‐I cells of rabbit carotid bodies were studied with the patch‐clamp technique in the whole‐cell and on the cell‐attached configuration. Cells exhibiting resting potentials of about −40 mV under normoxic conditions (P
O2: 20 kPa), depolarized during hypoxia (P
O2: 3.7 kPa). Hypoxia did not affect inward Ca2+ currents but inactivated outward K+ currents in voltage‐clamp experiments. Single‐channel currents recorded for the cell‐attached mode showed a slope conductance of about 137 pS and a 0 mV reversal potential under symmetrical K+ concentration (140 mM). The open‐probability (P
o) of the single channel was dependent on the extracellular P
O2. These data demonstrate the existence of a P
O2‐sensitive K+ channel in type‐I cells, which may account for cell depolarization and the resulting chemosensory response.
Cloning and sequencing of the human nucleolin cDNA
Authors: Srivastava Meera , Fleming Patrick J. , Pollard Harvey B. , Burns A.Lee
Published: July 2002

A cDNA containing the entire coding region for human nucleolin has been isolated from a λ gt10 human retinal library using a bovine cDNA probe. The cDNA hybridized to a transcript of 3000 bases from fast‐dividing cells, as well as terminally differentiated tissues of several species. Translation of the nucleotide sequence revealed a long open reading frame which predicts a 707 amino acid protein with several distinct domains. These include repeating elements, four conserved RNA‐binding regions, a glycine‐rich carboxy‐terminal domain and sites for phosphorylation, glycosylation and dibasic cleavage. Human and bovine nucleolin exhibited more additions and/or substitutions of aspartate, glutamate and serine residues in the chromatin‐binding domains by comparison with the hamster and mouse nucleolins. These differences may be related to species‐specific functions in transcription.
Isolation and structure of corazonin, a cardioactive peptide from the American cockroach
Authors: Veenstra Jan A.
Published: July 2002

Corazonin, a new cardioaccelerating peptide, has been isolated from the corpora cardiaca of the American cockroach, Periplaneta americana, and its structure determined to be Glp‐Thr‐Phe‐Gln‐Tyr‐Ser‐Arg‐Gly‐Trp‐Thr‐Asn‐amide. The peptide stimulated heart beat at concentrations as low as 0.2 nM, which makes it the most potent insect cardioactive neuropeptide.
Characterization of Mg2+ efflux from human, rat and chicken erythrocytes
Authors: Günther T. , Vormann J.
Published: July 2002

Net Mg2+ efflux from Mg2+‐loaded human, rat and chicken erythrocytes was measured in sucrose, NaCl and choline Cl medium. Thus, Na+‐dependent (NaCl minus choline Cl) and Na+‐independent Mg2+ efflux (in sucrose) were determined. Na+‐dependent Mg2+ efflux amounted to 0.16, 8.9 and 1.57 mmol/l cells × 30 min, Na+‐independent Mg2+ efflux amounted to 0.89, 1.55 and 0.37 mmol/l cells × 30 min for human, rat and chicken erythrocytes. Na+‐dependent Mg2+ efflux was inhibited by quinidine. Na+‐independent Mg2+ efflux was inhibited by SITS and Cl−. A small fraction of Na+‐independent Mg2+ efflux (in choline Cl) was resistant to SITS and Cl−. Ca2+ loading increased Mg2+ efflux similar to K+ efflux (Gardos effect). This effect was differently expressed in human and chicken erythrocytes.
An enzyme caught in action: Direct imaging of hydrolytic function and domain formation of phospholipase A2 in phosphatidylcholine monolayers
Authors: Grainger D.W. , Reichert A. , Ringsdorf H. , Salesse C.
Published: July 2002

Phospholipase A2, a ubiquitous lipolytic enzyme that actively catalyses hydrolysis of phospholipids, has been studied as a model for enzyme‐substrate reactions, as a membrane structural probe, and as a model for lipid‐protein interactions. Its mechanism of action remains largely controversial. We report here for the first time direct microscopic observation of the lipolytic action of fluorescently marked phospholipase A2 (Naja naja naja) against phosphatidylcholine monolayers in the lipid phase transition region. Under these conditions, phospholipase A2 is shown to target and hydrolyse solid‐phase lipid domains of L‐α‐dipalmitoylphosphatidylcholine. In addition, after a critical extent of monolayer hydrolysis, the enzyme itself aggregates into regular, visible proteinaceous domains within the lipid monolayer. Solid‐phase lipid hydrolysis indicates a preferential hydrolytic environment for phospholipase A2 while enzyme domain formation points to a possible allosteric inhibition mechanism by hydrolysis products.
The effect of heparin on the inositol 1,4,5‐trisphosphate receptor in rat liver microsomes Dependence on sulphate content and chain length
Authors: Tones Michael A. , Bootman Martin D. , Higgins Bernard F. , Lane David A. , Pay Graham F. , Lindahl Ulf
Published: July 2002

Heparin is known to inhibit the binding of inositol 1,4,5‐trisphosphate (Ins 1,4,5‐P3) to high‐affinity binding sites and to inhibit Ins 1,4,5‐P3‐induced Ca2+ release from intracellular membrane‐bound stores [(1987) J. Biol. Chem. 262, 12132–12136; (1987) FEBS Lett. 228, 57–59]. We have performed studies to clarify the structural requirements for this action of heparin in rat liver microsomes. Both N‐ and O‐linked sulphate groups contribute to binding activity, since de‐N‐sulphated heparin was without effect on the Ins 1,4,5‐P3 receptor whereas a polyxylan bearing only O‐linked sulphates (pentosan polysulphate) was as active as heparin. Therefore, the density of negative charge contributed by sulphate groups is important for the binding of heparin. Heparins with high and low affinity for antithrombin III both inhibited Ins 1,4,5‐P3 binding. There was a strong dependence on chain length, since binding activity decreased dramatically as the size of the heparin chain was reduced below that of 18–24 monosaccharide units.
The narK gene product participates in nitrate transport induced in Escherichia coli nitrate‐respiring cells
Authors: Noji Sumihare , Nohno Tsutomu , Saito Taiichi , Taniguchi Shigehiko
Published: July 2002

The nucleotide sequence of the Escherichia coli narK gene, which is located in the upstream region of the narCHJI operon, was determined. The narK gene encodes a very hydrophobic protein with 463 amino acid residues (M
r 49 693). A narK deletion mutant, under conditions for the induction of nitrate respiration, was unable to perform nitrate transport. Loss of transport activity was recovered by transforming the mutant with a narK
+ plasmid. Thus, we conclude that the narK gene encodes a transmembrane protein participating in nitrate transport. In the narK promoter region, we defined a unique sequence that we designate as a ‘nitrate box’, functioning as a putative NarL‐binding site, in addition to the consensus sequence of the ‘anaero‐box’.
A 16 kDa protein co‐isolating with gap junctions from brain tissue belonging to the class of proteolipids of the vacuolar H+‐ATPases
Authors: Dermietzel Rolf , Völker Michael , Hwang Theng-Khing , Berzborn Richard J. , Meyer Helmut E.
Published: July 2002

A 16 kDa protein from an enriched gap junction preparation was isolated from bovine brain tissues. N‐terminal amino acid microsequencing of the first 20 amino acids showed a complete homology with a recently published sequence of a proteolipid from a vacuolar H+‐ATPase from chromaffin granules. Incubation of the brain gap junction preparation with 14C‐N,N′‐dicyclohexylcarbodiimide showed a significant binding of this compound to the 16 kDa protein, indicating that a proton binding site also occurs within that particular protein. The data suggest that this 16 kDa protein, which has also been described in gap junction preparations from various other tissues, belongs to the proton transporting ATPase.
Linker histone‐DNA complexes: enhanced stability in the presence of aluminum lactate and implications for Alzheimer's disease
Authors: Lukiw W.J. , Kruck T.P.A. , McLachlan D.R.
Published: July 2002

The binding of human brain linker histone proteins to a radiolabelled human Alu repetitive element was examined by mobility shift assay.. Analysis of the complexes formed from protein extracts of whole neocortical nuclei, under physiological conditions in vitro revealed that linker histone H1° has the highest affinity for the Alu DNA sequence. The linker histone‐DNA complexes assembled in the presence of aluminum lactate were more resistant to sodium chloride‐induced dissociation than those formed in the presence of sodium lactate. The enhanced stability of deoxyribonucleoprotein (DNP) complexes in the presence of the aluminum cation may be of significance in neurodegenerative conditions such as Alzheimer's disease where aluminum preferentially associates with DNA containing structures of the nucleus.
Inhibition of the aspartic proteinase from HIV‐2
Authors: Richards Anthony D. , Broadhurst Anne V. , Ritchie Alison J. , Dunn Ben M. , Kay John
Published: July 2002

Kinetic constants were determined for the interaction of the HIV‐2 aspartic proteinase with a synthetic substrate and a number of inhibitors at several pH values. Acetyl‐pepstatin was more effective towards HIV‐2 proteinase than the renin inhibitor, H‐261; this effect is exactly the opposite from that observed previously for the proteinase from the HIV‐1 AIDS virus.
Nitrite reductase from Pseudomonas aeruginosa: Sequence of the gene and the protein
Authors: Silvestrini Maria Chiara , Galeotti Cesira L. , Gervais Michel , Schininà Eugenia , Barra Donatella , Bossa Francesco , Brunori Maurizio
Published: July 2002

The gene coding for nitrite reductase of Pseudomonas aeruginosa has been cloned and its sequence determined. The coding region is 1707 bp long and contains information for a polypeptide chain of 568 amino acids. The sequence of the mature protein has been confirmed independently by extensive amino acid sequencing. The amino‐terminus of the mature protein is located at Lys‐26; the preceding 25 residue long extension shows the features typical of signal peptides. Therefore the enzyme is probably secreted into the periplasmic space. The mature protein is made of 543 amino acid residues and has a molecular mass of 60204 Da. The c‐heme‐binding domain, which contains the only two Cys of the molecule, is located at the amino‐terminal region. Analysis of the protein sequence in terms of hydrophobicity profile gives results consistent with the fact that the enzyme is fully water soluble and not membrane bound; the most hydrophilic region appears to correspond to the c‐heme domain. Secondary structure predictions are in general agreement with previous analysis of circular dichroic data.
Tryptophan decarboxylase from Catharanthus roseus is a pyridoxoquinoprotein
Authors: Pennings Ed J.M. , Groen Barend W. , Duine Johannis A. , Verpoorte Robert
Published: July 2002

Tryptophan decarboxylase (EC 4.1.1.28) from Catharanthus roseus was purified to homogeneity. The native enzyme has an M
r of about 96000 as estimated from native PAGE. After SDS‐PAGE, three protein bands were visible corresponding with M
r 49000, 33000 and 17000. The N‐termini of the 49 kDa protein and the 33 kDa protein were identical. Antibodies against the 49 kDA protein also reacted strongly with the two smaller proteins. It is concluded that the native enzyme consists of two subunits of M
r 49000. Tryptophan decarboxylase appears to be a pyridoxo‐quinoprotein, since two molecules of pyridoxal phosphate and two molecules of covalently‐bound pyrroloquinoline quinone were found per enzyme molecule.
Messenger RNA encoding the D2 dopaminergic receptor detected by in situ hybridization histochemistry in rat brain
Authors: Najlerahim A. , Barton A.J.L. , Harrison P.J. , Heffernan J. , Pearson R.C.A.
Published: July 2002

A 30 base synthetic oligonucleotide probe was used to detect the mRNA encoding the rat D2 dopaminergic receptor. On Northern analysis, the probe identified a single species of mRNA of approximately 2.9 kb, present at highest levels in the striatum but also found in the brainstem, neocortex and diencephalon. On sections, neurons containing high levels of the mRNA were detected in the striatum, the substantia nigra pars compacta and the ventral tegmental area. Lower levels of signal were seen over neurons in the hypothalamus, the frontal neocortex, and the globus pallidus.
Nonenzymatic transformations of enzymatically generated N‐acetyldopamine quinone and isomeric dihydrocaffeiyl methyl amide quinone
Authors: Sugumaran Manickam , Semensi Victor , Dali Hemalata , Saul Steven
Published: July 2002

We have recently demonstrated that the side chain hydroxylation of N‐acetyldopamine and related compounds observed in several insects is caused by a two‐enzyme system catalyzing the initial oxidation of catecholamine derivatives and subsequent isomerization of the resultant quinones to isomeric quinone methides, which undergo rapid nonenzymatic hydration to yield the observed products [Saul, S.J. and Sugumaran, M. (1989) FEBS Lett. 249, 155–158]. During our studies on o‐quinone/p‐quinone methide tautomerase, we observed that quinone methides are also produced nonenzymatically slowly, under physiological conditions. The quinone methide derived from N‐acetyldopamine was hydrated to yield N‐acetylnorepinephrine as the stable product as originally shown by Senoh and Witkop [(1959) J. Am. Chem. Soc. 81, 6222–6231], while the isomeric quinone methide from dihydrocaffeiyl methylamide exhibited a new reaction to form caffeiyl amide as the stable product. The identity of this product was established by UV and IR spectral studies and by chemical synthesis. We could not find any evidence of intramolecular cyclization of N‐acetyldopamine quinone to iminochrome‐type compound(s). The importance of quinone methides in these reactions is discussed.
Resting plasma membrane potential of rat peritoneal mast cells is set predominantly by the sodium pump
Authors: Bronner C. , Mousli M. , Eleno N. , Landry Y.
Published: July 2002

Changes in the plasma membrane potential of two histanmine‐releasing cells, rat peritoneal mast cells and basophilic leukemia cells 2H3 (RBL 2H3), were recorded with the potential‐sensitive dye bis‐oxonol. For mast cells, the presence of ouabain or the absence of K+ increased the fluorescence intensity of bis‐oxonol; gramicidin had no effect. For RBL 2H3 cells, the presence of ouabain and the absence of K+ also increased bis‐oxonol fluorescence but gramicidin also increased it. These results show that the plasma membrane potential of RBL 2H3 cells is set, in part, by the acitivity of the Na+ pump and in part by the K+ conductance, while that of rat mast cells is set predominantly by the Na+ pump.
Identification of rho as a substrate for botulinum toxin C3‐catalyzed ADP‐ribosylation
Authors: Quilliam Lawrence A. , Lacal Juan-Carlos , Bokoch Gary M.
Published: July 2002

Recombinant Aplysia rho and a GTP‐binding protein purified from human neutrophil membranes (G22K) were ADP‐ribosylated by botulinum toxin C3 with stoichiometries of 0.8 and 0.6, respectively. Rho and G22K appeared to be different proteins since (i) rho migrated faster on polyacrylamide gels, (ii) unlike G22K, rho did not require the presence of cytosol to be ADP‐ribosylated, (iii) G22K was not recognized by an anti‐rho antiserum, and (iv) antibody 142‐24E05 recognized G22K effectively but only poorly cross reacted with rho. ADP‐ribosylation had no effect on the ability of rho to bind or hydrolyse GTP. Therefore, it appears that there are multiple botulinum toxin C3 substrates and that the toxin exerts its effects on cell function by a mechanism other than modulating the GTPase activity of rho.
Molecular cloning and sequence analysis of cDNA encoding δ4‐3‐ketosteroid 5β‐reductase of rat liver
Authors: Onishi Yoshiaki , Noshiro Mitsuhide , Shimosato Tsunehiro , Okuda Kyuichiro
Published: July 2002

A cDNA clone encoding δ4‐3‐ketosteroid 5β‐reductase was isolated from rat liver cDNA libraries using antibodies specific for the enzyme and oligonucleotides as probes. The cDNA contained 981‐base pair open reading frame encoding 327 amino acid residues (M, 37 376) and an unusually long 3′‐untranslated region rich in AT sequence in the total length of 3189 base pairs. The predicted amino acid sequence contains th sequences similar to the putative NADPH‐ and steroid‐binding regions.
The complete amino acid sequence of ribonuclease from the seeds of bitter gourd (Momordica charantia)
Authors: Ide Hiroyuki , Kimura Makoto , Arai Mariko , Funatsu Gunki
Published: July 2002

The complete amino acid sequence of ribonuclease (RNase MC) from the seeds of bitter gourd (Momordica charantia) has been determined. This has been achieved by the sequence analysis of peptides derived by enzymatic digestion with trypsin, lysylendopeptidase, and chymotrypsin, as well as by chemical cleavage with cyanogen bromide. The protein contains 191 amino acid residues and has a calculated molecular mass or 21 259 Da. Comparison of this sequence with sequences of the fungal RNases, RNase T2, and RNase Rh, revealed that there are highly conserved residues at positions 32–38 (TXHGLWP) and 81–92 (FWXHEWXKHGTC). Furthermore, the sequence of RNase MC was found to be homologous to those of Nicotiana alata
S‐glycoproteins involved in self‐incompatibility sharing 41% identical residues.
Protein kinase C dependent and independent activation of phospholipase A2under calcium ionophore (A23187) exposure in rabbit pulmonary arterial smooth muscle cells
Authors: Chakraborti Sajal , Michael John R. , Patra Samir K.
Published: July 2002

Exposure of rabbit pulmonary arterial smooth muscle cells to the calcium ionophore A23187, dose‐dependently stimulates arachidonic acid (AA) release and phospholipase A2(PLA2) activity. The protein kinase C (PKC) inhibitor, sphingosine does not prevents AA release and PLA2activity caused by low doses of A23187. In contrast, sphingosine markedly prevents AA release and PLA2activity caused by higher doses of A23187. PKC activity profile indicates that treatment of the cells with low doses of A23187 does not cause significant alteration of PKC translocation from cytosol to membrane whereas higher concentrations of the ionophore dose‐dependently enhance PKC translocation from Cytosol to membrane in the smooth muscle cells.
Reduction in cab and psb A RNA transcripts in response to supplementary ultraviolet‐B radiation
Authors: Jordan Brian R. , Chow W.S. , Strid Åke , Anderson Jan M.
Published: July 2002

The cab and psb A RNA transcript levels have been determined in Pisum sativum leaves exposed to supplementary ultraviolet‐B radiation. The nuclear‐encoded cab transcripts are reduced to low levels after only 4 h of UV‐B treatment and are undetectable after 3 days exposure. In contrast, the chloroplast‐encoded psb A transcript levels, although reduced, are present for at least 3 days. After short periods of UV‐B exposure (4 h or 8 h), followed by recovery under control conditions, cab RNA transcript levels had not recovered after 1 day, but were re‐established to ca. 60% of control levels after 2 more days. Increased irradiance during exposure to UV‐B reduced the effect upon cab transcripts, although the decrease was still substantial. These results indicate rapid changes in the cellular regulation of gene expression in response to supplementary UV‐B and suggest increased UV‐B radiation may have profound consequences for future productivity of sensitive crop species.
Reactivation of metal‐requiring apoenzymes by phytochelatin—metal complexes
Authors: Thumann Josef , Grill Erwin , Winnacker Ernst-L. , Zenk Meinhart H.
Published: July 2002

The enzymatically inactive, metal‐requiring apoforms of diamino oxidase and of carbonic anhydrase were reactivated by copper‐ and zinc—phytochelatin complexes, respectively. The level and the rate of reactivation effected by metal complexes consisting of poly(γ‐glutamylcysteinyl)glycine as well as by the respective free metal ion were compared. An efficient transfer of zinc and copper from phytochelatin‐complexes to apoenzymes was observed in vitro.
Antithrombin Cambridge II, 384 Ala to Ser Further evidence of the role of the reactive centre loop in the inhibitory function of the serpins
Authors: Perry D.J. , Daly M. , Harper P.L. , Tait R.C. , Price J. , Walker I.D. , Carrell R.W.
Published: July 2002

Four unrelated individuals have been identified with an identical antithrombin variant, associated in one of them with episodes of recurrent venous thromboses. In each case, the plasma antithrombin concentration was normal and the only functional abnormality was a minor but consistent decrease in the heparin‐induced thrombin inhibition suggesting a mutation at or near the reactive centre of the molecule. Amplification and direct sequencing of exon 6 showed a G→T mutation at nucleotide 1246, which corresponds to a substitution of a serine for an alanine at residue 384. This is one of a series of conserved alanines that form the stalk to the reactive centre loop. The observed changes in this variant are compatible with recent structural studies that infer that mobility of this stalk with partial re‐entry into the A‐sheet of the molecule is necessary for optimal inhibitory activity.
Tissue‐specific expression of Na,K‐ATPase β‐subunit Does β2 expression correlate with tumorigenesis?
Authors: Akopyanz N.S. , Broude N.E. , Bekman E.P. , Marzen E.O. , Sverdlov E.D.
Published: July 2002

We have found a substantial decrease in the level of Na,K‐ATPase β2‐subunit mRNA in xenografts of human renal, lung hepatocellular carcinomas in nude mice as compared with corresponding normal tissues, as well as in the neuroblastoma cell line as compared with the neuron primary cell culture. The level of β1 mRNA is decreased in kidney and lung tumor cells, but is unchanged in hepatocellular carcinoma. In the neuroblastoma cell line the level of β1 subunit mRNA was found to be higher then in neuron primary cell culture. The level of α1 mRNA in investigated tumors was the same as in normal tissues. These results may give evidence of the involvement of β2‐subunit in the process of tumorigenesis as was shown for some other adhesion molecules.
Ribonuclease activity and substrate preference of human eosinophil cationic protein (ECP)
Authors: Sorrentino Salvatore , Glitz Dohn G.
Published: July 2002

The eosinophil cationic protein (ECP), a potent helminthotoxin with considerable neurotoxic activity, was recently shown to also have ribonucleolytic activity. In this work the substrate preference of ECP ribonuclease action was studied in detail. With single‐stranded RNA or synthetic polyribonucleotide substrates ECP showed significant but low activity, 70‐ to 200‐fold less than that of bovine RNase A. ECP hydrolyzed RNA more rapidly than it did any synthetic polynucleotide. Poly(U) was degraded more rapidly than poly(C), and poly(A) and double‐stranded substrates were extremely resistant. Defined low molecular weight substrates in the form of the 16 dinucleoside phosphates (NpN′) and uridine and cytidine 2′, 3′‐cyclic phosphates were tested, and none showed hydrolysis by ECP at a significant rate. The results link ECP ribonucleolytic activity to the ‘non‐secretory’ liver‐type enzymes rather than to the ‘secretory’ pancreatic‐type RNases.
The amino acid sequence of rusticyanin isolated from Thiobacillus ferrooxidans
Authors: Yano Takahiro , Fukumori Yoshihiro , Yamanaka Tateo
Published: July 2002

The amino acid sequence of rusticyanin, a copper protein, purified from the iron‐oxidizing bacterium Thiobacillus ferrooxidans was determined. Rusticyanin contained 154 amino acid residues in a single polypeptide chain and its molecular weight was calculated to be about 16400 based on the amino acid sequence. The N‐terminal sequence up to the 20th residue of the protein apparently resembled those of Methylobacterium extorquens AMI amicyanin and poplar leaf plastocyanin rather than those of azurin family proteins. In the C‐terminal region of the sequence, rusticyanin had one cysteine, one histidine and one methionine which are conserved through many copper proteins. In the middle region of the sequence, rusticyanin was not similar to any other copper protein. The sequence nearby His84 of rusticyanin was similar to those of other copper proteins to some extent. However, Asn which follows His84 and is highly conserved in other copper proteins did not exist in rusticyanin. Therefore, it seemed difficult to conclude on the basis of the results obtained in the present study that His84 in rusticyanin was the fourth ligand to the copper atom.
Brain nitric oxide synthase is a biopterin‐ and flavin‐containing multi‐functional oxido‐reductase
Authors: Mayer Bernd , John Mathias , Heinzel Burghard , Werner Ernst R. , Wachter Helmut , Schultz Günter , Böhme Eycke
Published: July 2002

Brain nitric oxide synthase is a Ca2+/calmodulin‐regulated enzyme which converts L‐arginine into NO. Enzymatic activity of this enzyme essentially depends on NADPH and is stimulated by tetrahydrobiopterin (H4biopterin). We found that purified NO synthase contains enzyme‐bound H4 biopterin, explaining the enzymatic activity observed in the absence of added cofactor. Together with the finding that H4 biopterin was effective at substoichiometrical concentrations, these results indicate that NO synthase essentially depends on H4 biopterin as a cofactor which is recycled during enzymatic NO formation. We found that the purified enzyme also contains FAD, FMN and non‐heme iron in equimolar amounts and exhibits striking activities, including a Ca2+/calmodulin‐dependent NADPH oxidase activity, leading to the formation of hydrogen peroxide at suboptimal concentrations of L‐arginine or H4 biopterin.
cDNA sequence of a Drosophila melanogaster gene, Dfur1, encoding a protein structurally related to the subtilisin‐like proprotein processing enzyme furin
Authors: Roebroek Anton J.M. , Pauli Ilse G.L. , Zhang Young , van de Ven Wim J.M.
Published: July 2002

Screening a genomic library of Drosophila melanogaster DNA with a human fur cDNA probe resulted in the isolation of DNA clones that apparently belonged to two different DNA regions of the Drosophila genome. Subsequently, corresponding Drosophila cDNA clones were isolated. Nucleotide sequence analysis indicated that these cDNA clones originated from two different genes, which were called Dfur1 and Dfur2. From overlapping Dfur1 cDNA clones, a composite cDNA could be constructed and analysis of its nucleotide sequence revealed the coding sequence for a protein of 899 amino acid residues. This protein, designated Dfurin1, exhibited striking sequence homology to human furin and contained the same protein domains except for the cysteine‐rich region. Furthermore, unlike human furin. Dfurin1 possessed an extended amino‐terminal region in which a potential transmembrane anchor was present.
Strong expression of foreign genes following direct injection into fish muscle
Authors: Hansen Ekkehard , Fernandes Kenneth , Goldspink Geoffrey , Butterworth Peter , Umeda Patrick K. , Chang Kin-Chow
Published: July 2002

We report here for the first time direct injection of genes into fish muscle in vivo. Plasmids used contain either SV40 early promoter, rabbit β‐cardiac myosin heavy chain promoter, human MxA promoter of an artificial promoter, fused to a chloramphenicol acetyltransferase (CAT) or β‐galactosidase reporter gene. CAT assays revealed that most gene constructs were highly expressed. Histochemical analysis showed that β‐galactosidase was strongly expressed at the site of injection within muscle fibres. This method provides an excellent system for testing expression of gene constructs, including those of mammalian origin, in fish muscle in vivo and has the potential for fish vaccination.
Analysis of the 5′ non‐coding region of rat liver S‐adenosylmethionine synthetase mRNA and comparison of the Mr deduced from the cDNA sequence and the purified enzyme
Authors: Alvarez Luis , Asunción Miryam , Corrales Fernando , Pajares Maria A. , Mato José M.
Published: July 2002

A 3 kb cDNA coding for rat liver S‐adenosylmethionine (AdoMet) synthetase has been isolated. The M
r of the protein has been unequivocally determined by cDNA sequencing and enzyme purification on a thiopropyl‐Sepharose column. The length of the mRNA 5′ non‐coding region has been defined by primer‐extension analysis. The rat liver cloned cDNA has been also used to detect S‐adenosylmethionine synthetase mRNA in human liver.
Activation of c‐myc promoter by c‐myc protein in serum starved cells
Authors: Kitaura Hirotake , Galli Ivo , Taira Takahiro , Iguchi-Ariga Sanae M.M. , Ariga Hiroyoshi
Published: July 2002

The function of the c‐myc protein, the product of a proto oncogene, is not clearly understood although many reports, including ours, suggest that the c‐myc protein plays several roles in the regulation of transcription and DNA replication. Here we examined the effects of c‐myc protein on transcription from the c‐myc promoter, and by inference its role in auto‐regulation, after introducing into cultured cells a c‐myc expression vector and a CAT reporter gene linked to the promoter and upstream region of the human c‐myc gene. To minimize the effects of the endogenous c‐myc protein on the exogenously added CAT reporter gene, the transfected cells were treated under serum‐free conditions. The results show that CAT expression from the myc promoter increased in a dose‐dependent manner after addition of the c‐myc expression vector, and that it also required the presence of a c‐myc binding sequence previously identified 2 kb upstream from c‐myc's first exon. Moreover, the domains of the c‐myc protein important for transactivation were determined by use of various deletions mutants of c‐myc cDNA. The results showed that the N‐terminal portion in the c‐myc protein was necessary for transactivation beside the C‐terminal portion containing basic region, helix‐loop‐helix, and leucine zipper.
Endonuclease (R) subunits of type‐I and type‐III restriction‐modification enzymes contain a helicase‐like domain
Authors: Gorbalenya Alexander E. , Koonin Eugene V.
Published: July 2002

A statistically significant amino acid sequence similarity is demonstrated between the endonuclease (R) subunit of EcoK restriction‐modification (R‐M) enzyme, and RNA and DNA helicases of the so‐called ‘DEAD’ family. It is further shown that all three known sequences of R subunits of type‐I and type‐III R‐M enzymes contain the conserved amino acid sequence motifs typical of the previously described helicase superfamily II [(1989) Nucleic Acids Res. 17, 4713–4730]. A hypothesis is proposed that these enzymes may exert helicase activity possibly required for local unwinding of DNA in the cleavage sites.
Direct measurement of the equilibrium between glutathione and dithiothreitol by high performance liquid chromatography
Authors: Chau Mei-Hing , Nelson Jeffrey W.
Published: July 2002

The equilibrium constant between reduced glutathione (GSH), oxidized glutathione (GSSG), reduced dithiothreitol (DTTSH
SH), and oxidized dithiothreitol (DTTS
S) has been directly measured by high performance liquid chromatography analysis of equilibrium mixtures. The equilibrium constant at 25°C for the reaction GSSG + DTTSH
SH ⇌ 2GSH + S
S varies from approximately 200 M, below pH 8, to approximately 2800 M, above pH 11. The observed pH dependence is generally consistent with published values of acid dissociation constants of these thiols.
Interaction of mastoparan with the low molecular mass GTP‐binding proteins rho/rac
Authors: Koch Gertrud , Haberman Barbara , Mohr Christiane , Just Ingo , Aktories Klaus
Published: July 2002

Mastoparan, which has been shown to active G proteins [1], inhibits the ADP‐ribosylation of 20 kDa human platelet membrane proteins catalyzed by Clostridium botulinum exoenzyme C3 half‐maximally and maximally (90%) at 20 and 100 μM concentrations, respectively. Inhibition of ADP‐ribosylation was enhanced by GTP‐γS. Mastoparan increased GTP hydrolysis by porcine brain rho protein and stimulated GTP binding in a concentration dependent manner. The data suggest that mastoparan not only interacts with heterotrimeric G proteins but also with low molecular mass GTP‐binding proteins of the rho/rac family.
In vivo study of the state of order of the membranes of Gram‐negative bacteria by Fourier‐transform infrared spectroscopy (FT‐IR)
Authors: Schultz Christian , Naumann Dieter
Published: July 2002

Temperature‐induced order/disorder transition profiles were obtained from the membranes of intact Gram‐negative bacterial cells by FT‐IR analysis of the frequency shifts of the acyl chain methylene symmetric stretching band as a monitor. Cells grown at different temperatures yielded distinct transition profiles. At the individual growth temperatures, however, the nearly alike frequency values indicated a very similar ‘state of order’ of the bacterial membranes. The FT‐IR data were complemented by GC analysis of whole cell fatty acid composition. The FT‐IR data obtained in vivo gave direct evidence of the adaptation of the ‘state of order’ and ‘fluidity’ of bacterial membranes to varying growth temperatures.
1H,13C and 15N backbone assignments of cyclophilin when bound to cyclosporin A (CsA) and preliminary structural characterization of the CsA binding site
Authors: Neri Placido , Meadows Robert , Gemmecker Gerd , Olejniczak Edward , Nettesheim David , Logan Timothy , Simmer Robert , Helfrich Rosalind , Holzman Thomas , Severin Jean , Fesik Stephen
Published: July 2002

The backbone 1H, 13C and 15N chemical shifts of cyclophilin (CyP) when bound to cyclosporin A (CsA) have been assigned from heteronuclear two‐ and three‐dimensional NMR experiments involving selectively 15N‐ and uniformly 15N‐ and 15N,13C‐labeled cyclophilin. From an analysis of the 1H and 15N chemical shifts of CyP that change upon binding to CsA and from CyP/CsA NOEs, we have determined the regions of cyclophilin involved in binding to CsA.
Synthesis of α1‐microglobulin in cultured rat hepatocytes is stimulated by interleukin‐6, leukemia inhibitory factor, dexamethasone and retinoic acid
Authors: Pierzchalski Piotr , Rokita Hanna , Koj Aleksander , Fries Erik , Åkerström Bo
Published: July 2002

The secretion or α1‐microglobulin by primary cultures of rat hepatocytes was found to increase upon the addition of interleukin‐6 or leukemia inhibitory factor, two mediators of acute phase response. This stimulatory effect was further enhanced by dexamethasone. α1‐Microglobulin is synthesized as a precursor also containing bikunin, and the precursor protein is cleaved shortly berore secretion. Our results therefore suggest that both α1‐microglobulin and bikunin are acute phase reactants in rat hepatocytes. Furthermore, we found that retinoic acid, previously shown to be involved in the regulation of cell differentiation and development, also stimulated α1‐microglobulin synthesis. Only free, uncomplexed α1‐microglobulin (28,000 Da) was detected in the hopatocyte media, suggesting that the complex between α1‐microglobulin and α1‐inhibitor 3, found in rat serum, is formed outside the hepatocyte.
The first epidermal growth factor domain of human coagulation factor VII is essential for binding with tissue factor
Authors: Clarke Bryan J. , Ofosu Frederick A. , Sridhara Sampath , Bona Robert D. , Rickles Frederick R. , Blajchman Morris A.
Published: July 2002

The intrinsic pathway of coagulation is initiated when zymogen factor VII binds to its cell surface receptor tissue factor to form a catalytic binary complex. Both the activation of factor VII to factor VIIa and the expression of serine protease activity of factor VIIa are dependent on factor VII binding to tissue factor lipoprotein. To better understand the molecular basis of these rate‐limiting events, the interaction of zymogen factor VII and tissue factor was investigated using as probes both a murine monoclonal antibody and a monospecific rabbit antiserum to human factor VII. To measure factor VIIa functional activity, a two‐stage chromogenic assay was used; an assay which measures the factor Xa generated by the activation of factor VII to factor VIIa. Purified immunoglobulin from murine monoclonal antibody 231‐7, which was shown to be reactive with amino acid residues 51–88 of the first epidermal growth factor‐like (EGF) domain of human factor VII, inhibited the activation of factor VII to factor VIIa in a dose‐dependent manner. The mechanism of this inhibition was demonstrated using a novel solid‐phase ELISA which quantitatively measured the binding of purified factor VII zymogen to tissue factor adsorbed onto microtiter wells. Thus, the binding of factor VII zymogen to immobilized tissue factor was inhibited by antibody 231‐7, again in a dose‐dependent manner. Similar results were obtained using a monospecific rabbit antiserum to human factor VII which also reacted with the β‐galactosidase fusion proteins containing amino acid residues 51–88 (exon 4) of human factor VII. We conclude therefore that the exon 4‐encoded amino acids of the first EGF domain of human factor VII constitute an essential domain participating in the binding of factor VII to tissue factor.