bluuebunny/crossref_metadata_2025 huggingface dataset:
Preferential binding of collagenase to α2‐macroglobulin in the presence of the tissue inhibitor of metalloproteinases
Authors: Cawston Timothy E. , Mercer Elizabeth
Published: July 2002

The binding of collagenase to both α2‐niacroglobulin and the tissue inhibitor of metalloproteinases was studied using purified materials. Collagenase bound preferentially to α2‐macroglobulin although no transfer of collagenase to α2‐macroglobulin occurred if the enzyme was first mixed with the tissue inhibitor of metalloproteinases. The sequences of amino acids in both inhibitors likely to be responsible for the binding of collagenase are discussed and compared to the cleavage site in the collagen molecule.
Spacer alterations which increase the expression of porcine growth hormone inE. coli
Authors: Vize Peter D. , Wells Julian R.E.
Published: July 2002

Full‐length porcine growth hormone (PGH) cDNA clones were isolated from a porcine pituitary cDNA library. When the coding portion of the PGH gene was cloned into anE.coliexpression vector downstream from the powerfultrcpromoter, high levels of mRNA, but no protein were detected. Mutation directed by an oligodeoxynucleotide primer altered 5'‐non‐coding sequences and raised the level of PGH produced from undetectable to 15% of the total cellular protein. Alteration of four codons infrequently used byE.coliin the 5'‐end of the gene produced no further increases.
Ca2+ binding to the oxygen evolving enzyme varies with the redox state of the Mn cluster
Authors: Boussac Alain , Rutherford A.William
Published: July 2002

Oxygen evolution by the mangano‐enzyme of photosystem II is inhibited by Ca2+ depletion induced by NaCl washing and restored by Ca2+ addition. The effectiveness of NaCl treatment in inhibiting oxygen evolution in photosystem II was studied after a series of preilluminating flashes. The susceptibility of the enzyme to NaCl treatment varied with the number of preilluminating flashes and this variation showed an oscillation pattern with a period of four. This pattern is characteristic of cycling through the four long‐lived intermediate states in the enzyme cycle (i.e. the states, S0, S1, S2, S3). The relative extent of inhibition corresponding to each of the S states was as follows: S3 > S0≈S2 > S1. From these results it is concluded that Ca2+ binding is dependent on the S states and that Ca2+ probably plays a fundamental role in the mechanism of water splitting. The results also help to explain the conflicting reports of the extent of inhibition induced by NaCl washing and the controversy over which electron transfer step is inhibited by Ca2+ depletion.
Molecular cloning and sequence analysis of cDNA encoding human kidney D‐amino acid oxidase
Authors: Momoi Kyoko , Fukui Kiyoshi , Watanabe Fusao , Miyake Yoshihiro
Published: July 2002

cDNA clones encoding D‐amino acid oxidase were isolated from a human kidney cDNA library by hybridization with cDNA for the pig enzyme. The cDNA insert of 2.0 kilobase pairs long provided coding information for a protein consisting of 347 amino acids. The molecular mass of the enzyme was calculated to be 39 410 Da. The amino acid sequence similarity between the pig and human enzymes is 84.4%, and among the active site residues proposed from chemical modification studies, methionine‐110 of the pig enzyme was replaced by threonine. Northern blot analysis confirmed the expression of an mRNA of 2.0 kilobases encoding the D‐amino acid oxidase in human kidney.
Agonist‐stimulated high‐affinity GTPase in Dictyostelium membranes
Authors: Snaar-Jagalska B.Ewa , Jakobs Karl H. , Van Haastert Peter J.M.
Published: July 2002

GTP hydrolysis in Dictyostelium discoideum membranes is caused by a low (K
m> 1 mM) and a high affinity (K
m 6.5 μM) GTPase. cAMP enhances GTP hydrolysis apparently by increasing the affinity of the high affinity GTPase (stimulated K
m 4.5 μM); the low affinity GTPase was not affected by cAMP. Stimulation of GTP hydrolysis by cAMP was maximal at early time points and declined thereafter. A half‐maximal stimulation of GTPase occurred at 3 μM cAMP and the specificity of cAMP derivatives for stimulation of GTPase activity showed a close correlation with the specificity for binding to the cell surface cAMP receptor. Treatment of D. discoideum cells with pertussis toxin decreased the cAMP‐induced stimulation of GTPase from 42 ± 6% in control cells to 17 ± 9% in pertussis toxin‐treated cells. These results suggest that the interaction of cAMP with its surface receptor leads to stimulation of high affinity GTPase in D. discoideum membranes. At least one of those enzymes may represent a guanine nucleotide‐binding protein sensitive to pertussis toxin.
Do DNA polymerases δ and α act coordinately as leading and lagging strand replicases?
Authors: Focher Federico , Ferrari Elena , Spadari Silvio , Hübscher Ulrich
Published: July 2002

The activity ratio of DNA polymerases δ and α in calf thymus was found to be invariably 1:1, irrespective of extraction procedure (8 types) and subcellular localization (cytoplasm, nucleus and microsomes). This was established by separation of the two forms by hydroxyapatite chromatography and by their response to specific inhibitors and monoclonal antibodies. This finding supports the dimeric DNA polymerase model [(1980) J. Biol. Chem. 255, 4290–4303], which proposes that DNA polymerases δ and α act coordinately as leading and lagging strand enzymes, respectively, at the replication fork.
Methanobacterium thermoautotrophicum contains a soluble enzyme system that specifically catalyzes the reduction of the heterodisulfide of coenzyme M and 7‐mercaptoheptanoylthreonine phosphate with H2
Authors: Hedderich R. , Thauer R.K.
Published: July 2002

Cell extracts of Methanobacterium thermoautotrophicum (strain Marburg) were found to catalyze the reduction of the heterodisulfide of coenzyme M (CoM‐S‐H) and 7‐mercaptoheptanoylthreonine phosphate (H‐S‐HTP) with H2. All the activity was associated with the soluble cell fraction (160 000 × g supernatant). The enzyme system was purified sevenfold by anion‐exchange chromatography. The partially purified system had a specific activity of 100 nmol CoM‐S‐S‐HTP reduced per min and mg protein and exhibited an apparent K
m for CoM‐S‐S‐HTP of below 0.1 mM. The homodisulfides of CoM‐S‐H, of H‐S‐HTP, of cysteine, and of glutathione were not reduced. NADPH and NADH could not substitute for H2 as electron donor.
Silicomolybdate substitutes for the function of a primary electron acceptor and stabilizes charge separation in the photosystem II reaction center complex
Authors: Takahashi Yuichiro , Satoh Kimiyuki , Itoh Shigeru
Published: July 2002

Effects of silicomolybdate on the charge recombination between P680+ and the reduced pheophytin were studied by absorption and electron paramagnetic resonance spectroscopies in the photosystem II D1/D2/cytochrome b‐559 reaction center complex. This preparation lacks the primary and secondary quinone acceptors, QA and QB, and exhibits the charge recombination which produces the triplet state of P680 to a large extent. In the presence of silicomolybdate, the light‐induced triplet signal of P680 was almost completely eliminated at cryogenic temperatures as well as at 4°C. Under these conditions, two types of signals, one reversible and the other irreversible, which are ascribable to P680+ and the cation radical of antenna chlorophyll a, respectively, were generated upon illumination at cryogenic temperatures. These results indicate that silicomolybdate, which is known to be an artificial electron acceptor of QA, rapidly receives electrons from the reduced pheophytin even at cryogenic temperatures and thus suppresses the radical pair recombination which occurs in the time range of nanosecond. P680+ formed by flash excitation in the presence of silicomolybdate relaxed mainly with a long half decay time of 74 ms at 4°C. This indicates that the reduction of P680+ by the secondary electron donor, Z, is significantly decreased.
Catecholamines increase nerve growth factor mRNA content in both mouse astroglial cells and fibroblast cells
Authors: Furukawa Yoshiko , Tomioka Noboru , Sato Wakao , Satoyoshi Eijiro , Hayashi Kyozo , Furukawa Shoei
Published: July 2002

Previous studies have shown that catecholamines increase the nerve growth factor (NGF) content in medium conditioned by mouse L‐M fibroblast cells and mouse astroglial cells. In this study, the NGF mRNA levels in these cells were measured by Northern blot analysis. In astroglial cells treated with epinephrine (EN), the cellular NGF mRNA level increased prior to accumulation of NGF in the culture medium. 3‐Hydroxytyramine (DA) and norepinephrine (NE) also increased the cellular NGF mRNA content. An increased level of NGF mRNA elicited by EN was also observed in mouse L‐M cells. These results indicate that catecholamines enhance NGF synthesis of L‐M fibroblast cells and astroglial cells by increasing the cellular content of NGF mRNA. The present results also indicate that the effects of catecholamines are not mediated by adrenergic receptors.
A gene from pea (Pisum sativum L.) with homology to metallothionein genes
Authors: Marta Evans I. , Gatehouse Laurence N. , Gatehouse John A. , Robinson Nigel J. , Croy Ronald R.D.
Published: July 2002

While searching for ‘organ‐specific’ genes in pea (Pisum sativum L.) we have isolated a gene (designated PsMT
A) which has an ORF encoding a predicted protein with some similarity to metallothioneins (MTs). The PsMT
A transcript is abundant in roots which have not been exposed to elevated concentrations of trace metals.
Fast oxygen‐independent degradation of the DI reaction center protein in photosystem II
Authors: Jegerschöld Caroline , Styring Stenbjörn
Published: July 2002

The DI reaction center protein in photosystem II is rapidly degraded during illumination of chloride‐depleted or Tris‐washed thylakoids. The degradation is independent of oxygen and occurs under anacrobic conditions provided that electrons can flow through the acceptor‐side of photosystem II. This shows that oxygen‐derived reactive species are not necessarily involved in the light‐dependent damage of the DI protein. Instead the illumination of chloride‐depleted or Tris‐washed thylakoids induces long‐lived, strongly oxidizing radicals on the donor‐side of photosystems II which are suggested to be the damaging species for the DI protein.
A method for testing the stability of a steady‐state system during the calculation of a response to large changes in regulator concentrations
Authors: Crabtree B. , Newsholme E.A.
Published: July 2002

A modification of the ‘finite decomposition’ method (Crabtree and Newsholme (1985) Curr. Top. Cell. Regul. 25, 21–76) for calculating physiological responses from sensitivities is described, to enable the system to be tested for stability at each step of the procedure. Instability is indicated by a change of sign of the determinant of the square matrix (N) in the governing equation for the system. The method cannot be used to predict responses beyond any step at which instability occurs.
Recombinant poliovirus 3C protease The enzyme application to protein specific fragmentation
Authors: Sablina E.P. , Antonov V.K.
Published: July 2002

Active 3C protease of poliovirus I(M) was obtained when cloning and expressing fragment HindII‐HindIII (bases from 5240 to 6056) of cDNA in vector pTTQ8 in E.coli cells. As shown, fragment 3D of polymerase covalently bound to 3C does not deprive the enzyme of its specific proteolytic activity. The absence of 26 N‐terminal amino acids in 3C entails its inactivation. The recombinant 3C protease cleaved peptide bond Gln‐Gly not only in virus polyprotein, but also in molecules of β‐galactosidase and bovine catalase.
The density of ryanodine receptors decreases with pressure overload‐induced rat cardiac hypertrophy
Authors: Naudin V. , Oliviero P. , Rannou F. , Beuve C.Sainte , Charlemagne D.
Published: July 2002

We investigate the possibility that alterations in the calcium movements of the hypertrophied rat heart might involve sarcoplasmic reticulum (SR) ryanodine receptors. A decreased receptor density was observed with severe hypertrophy (0.26±0.05 and 0.35±0.06 pmol/mg protein and 170 and 366 receptors/μm2 of SR in 50–80% hypertrophy and control, respectively); however, the total number of receptors per left ventricle was unchanged. The dissociation constant (0.7 nM) was similar in both hypertrophied and control left ventricles. Thus the decreased density of the ryanodine receptors may participate in altered calcium movements in hypertrophied rat heart.
Inhibition of cell growth and tumorigenesis of human glioblastoma cells by a neutralizing antibody against human basic fibroblast growth factor
Authors: Takahashi Jun A. , Fukumoto Manabu , Kozai Yoshio , Ito Nobuyuki , Oda Yoshifumi , Kikuchi Haruhiko , Hatanaka Masakazu
Published: July 2002

We report here that a neutralizing mouse monoclonal antibody against basic FGF inhibited both anchorage‐dependent and anchorage‐independent growth of U‐87MG and T98G human glioblastoma cells and HeLa cells, all of which express both the basic FGF and the FGF remptor genes. In addition, the subcutaneous administration of this antibody significantly suppressed the tumor development or these tumor cells in nude mice. Therefore, basic FGF plays an important role in neoplastic growth of these cells. The neutralization or basic FGF will be effective in controlling the growth of tumors, such as glioblastoma and other cancer cells which bear basic FGF and FGF receptors.
A large conductance ion channel in the nuclear envelope of a higher plant cell
Authors: Matzke A.J.M. , Behensky C. , Weiger T. , Matzke M.A.
Published: July 2002

To detect and characterize ion channel activity in the nuclear envelope of a higher plant cell, we performed patch clamp experiments on nuclei isolated from coconut endosperm cells and on giant liposomes containing nuclear envelope fragments prepared from the same cells. An ion channel exhibiting a number of conductance substates, with a maximum of ca. 1,000 pS, was observed. Above an applied potential of ± 100 mV, the behavior of the channel was similar in isolated nuclei and liposomes, indicating that both patch clamp modes were detecting the same channel. That such a channel has now been identified in members of both the animal and plant kingdoms reinforces the notion that the nuclear pores are not always open to ions.
Insulin‐like growth factor II stimulates glucose transport in human skeletal muscle
Authors: Zierath Juleen R. , Bang Peter , Galuska Dana , Hall Kerstin , Wallberg-Henriksson Harriet
Published: July 2002

We investigated the effect of insulin‐like growth factor II (IGF‐II) and insulin‐like growth factor binding protein‐1 (IGFBP‐1) on 3‐O‐methylglucose transport in incubated human skeletal muscle strips. Increasing physiological concentrations of IGF‐II stimulated glucose transport in a dose‐dependent manner. Glucose transport was maximally stimulated in the presence of 100 ng/ml (13.4 nM) of IGF‐II, which corresponded to the effect obtained by 100μU/ml (0.6 nM) of insulin. Exposure of muscle strips to IGFBP‐1 (500 ng/ml) inhibited the maximal effect of IGF‐II on glucose transport by 40%. Thus, it is conceivable that IGF‐II and IGFBP‐I are physiological regulators of the glucose transport process in human skeletal muscle.
Characterization of a 41 kDa photoinhibition adduct in isolated photosystem II reaction centres
Authors: Barbato Roberto , Friso Giulia , Rigoni Fernanda , Frizzo Annalisa , Giacometti Giorgio M.
Published: July 2002

When isolated reaction centres of photosystem II are subjected to photoinhibitory illumination, a 41 kDa SDS‐PAGE band is observed under all experimental conditions. The same band is also found, together will lower molecular weight fragments of the D1 protein, in whole thylakoids and in all PSII sub‐particles investigated up to now. In the case of isolated reaction centres the 41 kDa band is represented by a heterodimer of the D1 polypeptide and the α‐subunit of cytochrome b
559. The cross‐linkage between D1 and α‐cyl b
559 involves a region on D1 between the N‐terminal residue and Arg·225, and is an early event in photo‐induced damage to the D1 protein.
The two Staphylococcal bi‐component toxins, leukocidin and gamma‐hemolysin, share one component in common
Authors: Kamio Yoshiyuki , Rahman Arifur , Nariya Hirofumi , Ozawa Toshiko , Izaki Kazuo
Published: July 2002

Staphylococcal bi‐component toxins, leukocidin and γ‐hemolysin, consist of two protein components, i.e. F and S for leukocidin and HγI and HγII for γ‐hemolysin. In this study we purified HγI and HγII to homogeneity from the culture medium of Staphylococcus aureus RIMD 310925 and compared their properties with those of F and S purified from the same source. The N‐terminal 59‐ and C‐terminal 2‐residue amino acid sequences, apparent molecular mass, and isoelectri point of purified HγI were the same as those of F. In an Ouchterlony double diffusion test a fused line without spur was formed between F and HγI using either anti‐F or anti‐HγI antibodies. A synergistic action of F and HγII caused hemolysis of human red blood cells, and HγI acted synergistically with S to exhibit leukocidin activity. We conclude that the two toxins share one protein component (F = HγI) in common and leukocidin‐ and γ‐hemolysin‐specific activities are determined by S and HγII, respectively. It is also reported that the N‐terminal 58‐residue sequence of HγII is 72% similar to the corresponding sequence of S.
In vitro phosphorylation of purified tobacco‐leaf phosphoenolpyruvate carboxylase
Authors: Wang Yue-Hao , Chollet Raymond
Published: July 2002

C3‐leaf phosphoenolpyruvate (PEP) carboxylase (PEPC) was purified about 1,000‐fold from tobacco and displayed a final specific activity of 35 μmol/min/mg protein, an apparent K
m (total PEP) of 95 mM (both at (pH 8.0, 30°C), and an I50(l‐malate) value of 0.14 mM at pH 7.3, 0.2 mM PEP. The rapid, 5‐step protocol involved polyethylene glycol fractionation and sequential FPLC on hydroxylapatite, phenyl‐Sepharose, Mono Q and Superose 12. The electrophoretically pure protein and purified C4‐leaf PEPC were phosphorylated in vitro in a reconstituted system with PEPC‐kinase isolated from illuminated tobacco and maize leaves. These reciprocal phosphorylation experiments (i) indicate that Ser11 of tobacco PEPC is the likely target residue, situated in the plant‐invariant Glu/Asp‐Lys/Arg‐X‐X‐Ser phosphorylation motif near the N‐terminus, and (ii) lend support to the recent hypothesis that C3‐leaf PEPC is subject to regulatory phosphorylation in vivo.
Expression of a pokeweed antiviral protein in Escherichia coli and its characterization
Authors: Kataoka Jiro , Ago Hideo , Habuka Noriyuki , Furuno Masahiro , Masuta Chikara , Miyano Masashi , Koiwai Akira
Published: July 2002

Two expression vectors were constructed to produce a putative mature α‐pokeweed antiviral protein (α‐PAP) in Escherichia coli with its NH2‐ and COOH‐terminal extrapeptides excised. One was for its intracellular expression with a methionine at its NH2‐terminal. The other was for its secretion using an ompA signal peptide. The former product was purified from the total soluble proteins of the transformant with a yield of 1.74 mg/liter and the latter had a yield of 5.55 mg/liter. Both products exhibited RNA N‐glycosidase activity on wheat ribosomes and inhibitory activity to protein synthesis in a rabbit reticulocyte system.
Chloroplast thylakoid protein phosphatase reactions are redox‐independent and kinetically heterogeneous
Authors: Silverstein Todd , Cheng Lüling , Allen John F.
Published: July 2002

At least eleven thylakoid proteins become phosphorylated under reducing conditions, and redox titration has identified a common midpoint potential of E
m = +38 ± 4 mV, n = 0.95 ± 0.06. In the presence of the phosphatase inhibitor NaF (10 mM), the redox dependency of phosphorylation is found to be essentially unchanged: E
m = +50 ± 3 mV, n = 1.02 ± 0.04. Thylakoid membranes were phosphorylated in the light and then incubated at various redox potentials for 15 min in the dark; no redox dependency was observed in the dephosphorylation of any of the 17 bands then distinguishable by autoradiography and phosphorimaging. The phosphoprotein phosphatase reactions can be divided arbitrarily into four kinetic classes: the fastest, class I, includes LHC II; the moderate class II includes D1 and D2; the slow class III includes CP43 and the 9 kDa phosphoprotein; finally, a 19.5 kDa protein exhibited no loss of32P at all. In separate experiments we measured thylakoid protein dephosphorylation initiated by changing the redox potential from −140 to +200 mV, in the presence or absence of fluoride. In this case the results are consistent with at least two kinetically distinguishable classes of phosphoprotein phosphatase reactions. We conclude that thylakoid protein phosphatase reactions are kinetically heterogeneous and redox‐independent. It follows that the redox dependency of thylakoid protein phosphorylation is a property of thylakoid protein kinase reactions. Our observed E
m and n values are consistent with a primary site of kinase redox control at the level of PQ/PQ.− of the Qi (Qn) site of the cytochrome b
6/ƒ complex.
The βA4 amyloid precursor protein binding to copper
Authors: Hesse Lars , Beher Dirk , Masters Colin L. , Multhaup Gerd
Published: July 2002

Previously it has been shown that the extracellular domain of transmembrane βA4 amyloid precursor protein (APP) includes binding sites for zine(II) and for molecules of the extracellular matrix such as collagen, laminin and the heparin sulfate chains of proteoglycans (HSPGs). Here we report that APP also binds copper ions. A copper type II binding site was located within residues 135–155 of the cysteine‐rich domain of APP695 which is present in all eight APP splice isoforms known so far. The two essential histidines in the type II copper binding site of APP are conserved in the related protein APLP2. Copper(II) binding is shown to inhibit homophilic APP binding. The identification of a copper(II) binding site in APP suggests that APP and APLP2 may be involved in electron transfer and radical reactions.
Functional alterations of the mitochondrially encoded ND4 subunit associated with Leber's hereditary optic neuropathy
Authors: Esposti Mauro Degli , Carelli Valerio , Ghelli Anna , Ratta Marina , Crimi Massimo , Sangiorgi Simonetta , Montagna Pasquale , Lenaz Giorgio , Lugaresi Elio , Cortelli Pietro
Published: July 2002

Leber's hereditary optic neuropathy (LHON) is a maternally inherited disease associated with point mutations in mitochondrial DNA. The most frequent of these mutations is the G‐to‐A substitution at nucleotide position 11,778 which changes an evolutionarily conserved arginine with a histidine at position 340 in subunit ND4 of NADH: ubiquinone reductase (respiratory complex I). We report that this amino acid substitution alters the affinity of complex I for the ubiquinone substrate and induces resistance towards its potent inhibitor rotenone in mitochondria of LHON patients. Such changes could reflect a substantial loss in the energy conserving function of NADH: ubiquinone reductase and thus explain the pathological effect of the ND4/11,778 mutation.
Calcium channel antagonists induce direct inhibition of the outward rectifying potassium channel in tobacco protoplasts
Authors: Thomine Sébastien , Zimmermann Sabine , Van Duijn Bert , Barbier-Brygoo Hélène , Guern Jean
Published: July 2002

Verapamil, nifedipine and bepridil, three antagonists of L‐type calcium channels in animal cells, were shown to induce direct inhibition of outward rectifying potassium current in Nicotiana tabacumcv. Xanthi protoplasts with IC50 of 5 μM, 5 μM and 1 μM, respectively. In the outside‐out configuration, verapamil reduced the open probability of the ion channel responsible for the outward rectifying potassium conductance. Verapamil also blocked the outward rectifying potassium conductance in protoplasts from the N. tabacum cv. Bright Yellow cell line. Thus, studies using these molecules to demonstrate the involvement of calcium channels in plant physiological responses should be regarded with caution.
Replication of adenovirus DNA in vitro is ATP‐independent
Authors: Pronk Rob , Van Driel Wim , Van der Vliet Peter C.
Published: July 2002

Using a reconstituted system for adenovirus DNA replication we tested the requirements for ATP and divalent cations. At the standard Mg2+ concentration ATP stimulated initiation 5 to 10‐fold. However, this effect was caused by complexing Mg2+. At the optimal Mg2+ concentration ATP was not required for initiation or elongation. Besides Mg2+ also Mn2+, Ca2+ and Ba2+ were shown to support initiation whereas for elongation only Mg2+ was accepted. Since Mn2+could efficiently be used for DNA chain elongation on synthetic templates we hypothesize that Mg2+ is essential for the transition of initiation to elongation.
Dictyostelium discoideum contains a family of calmodulin‐related EF‐hand proteins that are developmentally regulated
Authors: André Bernadette , Noegel Angelika A. , Schleicher Michael
Published: July 2002

A full‐length genomic DNA fragment that codes for a novel EF‐hand protein from Dictyostelium discoideum was cloned and sequenced. The protein is composed of 168 amino acids and contains four consensus sequences that are typical for Ca2+‐binding EF‐hand domains. The protein sequence exhibits only minor similarities to other calmodulin‐type proteins from Dictyostelium. The genomic DNA harbors two short introns; their positions suggest that the gene is unrelated to the EF‐hand proteins from the calmodulin group. Northern blot analysis showed that the mRNA level was significantly increased during development. Polyclonal antibodies raised against the recombinant protein recognized in Western blots a protein of about 20 kDa. Like the mRNA, also the protein was more abundant in developing cells. Overlay experiments with 45Ca2+ indicated that the EF‐hands in fact have Ca2+‐binding activity. The recent description of CBP1, another calmodulin‐type Dictyostelium protein that is upregulated during development [Coukell et al. (1995) FEBS Lett. 362, 342–346], suggests that D. discoideum contains a family of EF‐hand proteins that have specific functions during distinct steps of development. We therefore designate the protein described in this report as CBP2.
Novel anti‐platelet aggregation polypeptides from Vipera lebetina venom: Isolation and characterization
Authors: Barbouche Rym , Marrakchi Neziha , Mansuelle Pascal , Krifi Mohamed , Fenouillet Emmanuel , Rochat Hervé , El Ayeb Mohamed
Published: July 2002

Lebetins 1 and Lebetins 2, two polypeptide groups that inhibit platelet aggregation, were isolated from Vipera lebetina venom by gel filtration and reverse phase chromatography. Amino acid sequencing indicated that the first group contains two major polypeptides of 13 and 12 residues; their molecular weight was determined by electrospray mass spectrometry. The second was composed of two peptides of 38 and 37 residues, each with one disulfide bond. Sequence analysis revealed neither RGD sequence nor homology with other proteins including known snake or tick polypeptides. Lebetins 1 were Pro and Lys rich peptides and their sequences were identical to the N‐terminus of Lebetins 2. Lebetins inhibited platelet aggregation induced by thrombin, collagen and PAF‐acether. The 50% concentration that inhibited human and rabbit platelet aggregation induced by thrombin was 590 nM and 125 nM for Lebetins 1 and 100 nM and 8 nM for Lebetins 2, respectively. Lebetins 1 and Lebetins 2 also inhibited fibrinogen‐induced aggregation of α‐chymotrypsin‐treated platelets as well as in vivo collagen‐induced thrombocytopenia in rats with half effective doses of 2 nmol/kg and 4.2 nmol/kg, respectively. Lebetins were not toxic after intravenous injection into mice and rats. These polypeptides form novel platelet inhibitors that could be used to delineate further the mechanisms of platelet aggregation and serve as a model for developing antithrombotic agents.
A comparative investigation of hierarchical clustering techniques and dissimilarity measures applied to the cell formation problem
Authors: Vakharia Asoo J. , Wemmerlöv Urban
Published: July 2002

One methodology for identifying alternative cell designs is the use of clustering algorithms coupled with dissimilarity measure. This paper investigates the performance of seven hierarchical clustering techniques (six previously developed and one developed specifically for cell formation) and eight dissimilarity measures (three well‐known measures and five versions of a recently developed parametric measure) in the context of cell formation. Twenty‐four data sets, at close to 200 partition levels, and ten measures of performance, are used for this purpose. We first identify clustering techniques and dissimilarity measures which should not be used for cell formation when binary data are involved. From the remaining clustering techniques and dissimilarity measures, we then identify clustering technique/dissimilarity measure combinations which are consistently good or poor performers when cell characteristics are observed. High internal cell cohesiveness and low levels of machine duplication are shown to be conflicting goals. Clustering techniques' performance dependency on dissimilarity measures, data sets, stopping rules, and metrics are also clearly illustrated. Another result is that choice of clustering technique is more critical than choice of dissimilarity measure. However, differences among clustering techniques (due to chaining tendencies) can be sharply reduced by restricting the solution space for acceptable cell configurations.
Effects of hypnotics on sleep quality and daytime well-being. Data from a comparative multicentre study in outpatients with insomnia
Authors: Hajak G , Clarenbach P , Fischer W , Haase W , Bandelow B , Adler L , Rüther E
Published: July 2002

The effect of treatment (28 days) with zopiclone, triazolam, flunitrazepam and placebo on sleep quality and daytime well-being was proven in a randomised, double-blind, parallel group, multicentre study in private practice. Results of an exploratory statistic of treatment efficacy in a subgroup of 1,291 patients suffering from insomnia are presented. Patients met the following criteria: insomnia lasting at least four weeks and the presence of at least two of the following: 1) sleep latency ≥ 45 minutes, 2) total sleep time ≤ 6 hours, and 3) nocturnal awakening ≥3 times. Treatment efficacy was assessed according to the following factors: either a shortening of sleep latency by at least 15 minutes, or prolongation of total sleep time by at least 20%, or reduction of the number of nocturnal awakenings to three or less and a refreshed feeling in the morning as well as no impairment in daytime well-being due to tiredness or anxiety. The total response rate was markedly higher with zopiclone (42.3%; p = 0.0003) than with placebo (29.0%). Triazolam (36.6%; p = 0.0905) and flunitrazepam (33.1%; p = 0.3401) were also more effective than the placebo, but they both tended to have a lower response rate than with zopiclone (p = 0.1199 and 0.0151, respectively). Total response was found to be essentially a reflection of the response of the socially important parameter of daytime well-being. These results suggest that zopiclone is more effective in the treatment of insomnia than either triazolam or flunitrazepam. Since the response of daytime well-being to therapy was generally poor, this parameter embodies the next main therapeutic challenge in the treatment of patients with insomnia.
DNA stability in plant tissues: implications for the possible transfer of genes from genetically modified food
Authors: Chiter Amar , Forbes J.Michael , Blair G.Eric
Published: July 2002

The potential for transfer of antibiotic resistance genes from genetically modified (GM) plant material to microbes through genetic recombination in the human or animal gut is a consideration that has engendered caution in the use of GM foods. This study was aimed at defining the optimal physical and chemical conditions necessary to ensure sufficient fragmentation of DNA in plant tissues to a size where it would be unlikely to be stably transferred to bacterial gut microflora. The ribulose 1,5‐bisphosphate carboxylase/oxygenase small subunit (Rubisco SS) genes are of similar size (approximately 1.4 kb) to transgenes present in GM plants. DNA analysis and PCR amplification of Rubisco SS genes showed that fresh maize and maize silage contained high molecular weight DNA and intact Rubisco SS genes. Relatively high temperatures and pressurised steam were necessary to degrade fully genomic DNA and Rubisco SS genes in maize and wheat grains, the source of most animal feedstuffs. Furthermore, chemical expulsion and extrusion of oilseeds resulted in residues with completely degraded genomic DNA. These results imply that stringent conditions are needed in the processing of GM plant tissues for feedstuffs to eliminate the possibility of transmission of transgenes.
Identification of further important residues within the Glut4 carboxy‐terminal tail which regulate subcellular trafficking
Authors: Cope Diane L , Lee Shu-Hua , Melvin Derek R , Gould Gwyn W
Published: July 2002

The insulin‐responsive glucose transporter, Glut4, exhibits a unique subcellular distribution such that in the absence of insulin >95% of the protein is stored within intracellular membranes. In response to insulin, Glut4 exhibits a large mobilisation to the plasma membrane. Studies of the amino acid motifs which regulate the unique trafficking of Glut4 have identified several key residues within the soluble cytoplasmic N‐ and C‐terminal domains of Glut4. Of particular note is a Leu‐498Leu‐499 motif within the C‐terminal domain that has been proposed to regulate both internalisation from the plasma membrane and sorting to an insulin‐sensitive compartment. In this study, we have examined the role of the adjacent amino acids (Glu‐491, Gln‐492 and Glu‐493) by their sequential replacement with Ala. Our results are consistent with the notion that Glu‐491 and Glu‐493 play an important role in the sub‐endosomal trafficking of Glut4, as substitution of these residues with Ala results in increased levels of these proteins at the cell surface, reduced insulin‐stimulated translocation and increased susceptibility to endosomal ablation. These residues, together with other identified sequences within the C‐terminus of Glut4, are likely to be crucial targeting elements that regulate Glut4 subcellular distribution.
Activation of vanadium nitrogenase expression in Azotobacter vinelandii DJ54 revertant in the presence of molybdenum
Authors: Lei Shi , Pulakat Lakshmidevi , Gavini Nara
Published: July 2002

Azotobacter vinelandii carries three different and genetically distinct nitrogenase systems on its chromosome. Expression of all three nitrogenases is repressed by high concentrations of fixed nitrogen. Expression of individual nitrogenase systems is under the control of specific metal availability. We have isolated a novel type of A. vinelandii DJ54 revertant, designated A. vinelandii BG54, which carries a defined deletion in the nifH gene and is capable of diazotrophic growth in the presence of molybdenum. Inactivation of nifDK has no effect on growth of this mutant strain in nitrogen‐free medium suggesting that products of the nif system are not involved in supporting diazotrophic growth of A. vinelandii BG54. Similar to the wild type, A. vinelandii BG54 is also sensitive to 1 mM tungsten. Tn5‐B21 mutagenesis to inactivate the genes specific to individual systems revealed that the structural genes for vnf nitrogenase are required for diazotrophic growth of A. vinelandii BG54. Analysis of promoter activity of different nif systems revealed that the vnf promoter is activated in A. vinelandii BG54 in the presence of molybdenum. Based on these data we conclude that A. vinelandii BG54 strain utilizes vnf nitrogenase proteins to support its diazotrophic growth.
The first two N‐terminal immunoglobulin‐like domains of soluble human IL‐1 receptor type II are sufficient to bind and neutralize IL‐1β
Authors: Kollewe Christian , Neumann Detlef , Martin Michael U
Published: July 2002

Two forms of soluble human type II interleukin (IL)‐1 receptor (shIL‐1RII) were generated, one consisting of the complete extracellular three immunoglobulin (Ig)‐like domains and one containing only the first two N‐terminal Ig‐like domains. Both forms bound IL‐1β with a dissociation constant (K
d) of 200 pM and neutralized IL‐1β in a bioassay. They did not bind or neutralize IL‐1α. This demonstrates that the two Ig‐like domains of shIL‐1RII are sufficient to bind IL‐1β with an affinity comparable to full length shIL‐1RII. This suggests that this short form of shIL‐1RII contributes to the anti‐inflammatory effect of soluble IL‐1 receptors in vivo.
NAD+ analogs substituted in the purine base as substrates for poly(ADP‐ribosyl) transferase
Authors: Oei Shiao Li , Griesenbeck Joachim , Buchlow Gerhard , Jorcke Dierk , Mayer-Kuckuk Philipp , Wons Thomas , Ziegler Mathias
Published: July 2002

Poly(ADP‐ribosyl) transferase (pADPRT) catalyzes the transfer of the ADP‐ribose moiety from NAD+ onto proteins as well as onto protein‐bound ADP‐ribose. As a result, protein‐bound polymers of ADP‐ribose are formed. pADPRT itself contains several acceptor sites for ADP‐ribose polymers and may attach polymers to itself (automodification). In this study the influence of substitutions in the purine base of NAD+ on the polymerization reaction was investigated. The adenine moiety of NAD+ was replaced by either guanine, hypoxanthine or 1,N
6‐ethenoadenine. These analogs served as substrates for polymer synthesis as judged from the extent of automodification of the enzyme and the sizes of the polymers formed. Time course experiments revealed that 1,N
6‐etheno NAD+ (ϵ‐NAD+) and nicotinamide hypoxanthine dinucleotide (NHD+) were rather poor substrates as compared to NAD+. Synthesis of GDP‐ribose polymers from nicotinamide guanine dinucleotide (NGD+) was more efficient, but still significantly slower than poly(ADP‐ribosyl)ation of the enzyme using NAD+. The size of the different polymers appeared to correlate with these observations. After 30 min of incubation in the presence of 1 mM substrate, polymers formed from ϵ‐NAD+ or NHD+ contained up to 30 ϵ‐ADP‐ribose or IDP‐ribose units, respectively. Using NGD+ as substrate polymers consisted of more than 60 GDP‐ribose units, an amount similar to that achieved by poly(ADP‐ribosyl)ation in the presence of only 0.1 mM NAD+ as substrate. These results suggest that the presence of an amino group in the purine base of NAD+ may facilitate catalysis. Substitution of the nicotinamide moiety of NAD+ with 3‐acetylpyridine had no detectable effect on polymer formation. Oligomers of GDP‐ribose and ϵ‐ADP‐ribose exhibited a slower mobility in polyacrylamide gels as compared to ADP‐ribose or IDP‐ribose oligomers. This feature of the two former analogs as well as their markedly attenuated polymerization by pADPRT provide valuable tools for the investigation of the enzymatic mechanism of this protein. Moreover, polymers of ϵ‐ADP‐ribose may be useful for studying enzymes degrading poly(ADP‐ribose) owing to the fluorescence of this analog. Digestion of ϵ‐ADPR polymers with snake venom phosphodiesterase was accompanied by a significant fluorescence enhancement.
Ozone depletes tocopherols and tocotrienols topically applied to murine skin
Authors: Thiele Jens J , Traber Maret G , Podda Maurizio , Tsang Kenneth , Cross Carroll E , Packer Lester
Published: July 2002

To evaluate ozone damage to hairless mouse skin, two parameters of oxidative damage, vitamin E depletion and malondialdehyde (MDA) production, were measured in vitamin E‐enriched and in control skin from mice exposed to ozone (10 ppm). A 5% vitamin E solution (tocotrienol‐rich fraction, TRF) in polyethylene glycol (PEG) was applied to 2 sites on the back of hairless mice, PEG to 2 sites. After 2 h, the sites were washed, one of each pair of sites covered and the mice exposed ozone for 2 h. Ozone exposure (compared with covered sites) increased epidermal MDA in PEG‐treated sites, while vitamin E was unchanged. In contrast, ozone exposure significantly depleted vitamin E in TRF‐treated sites, while significant MDA accumulation was prevented. This is the first demonstration that ozone exposure causes damage to cutaneous lipids, an effect which can be attenuated by vitamin E application.
Nucleotide occupancy of F1‐ATPase catalytic sites under crystallization conditions
Authors: Löbau Sabine , Weber Joachim , Senior Alan E
Published: July 2002

© 1997 Federation of European Biochemical Societies.
cAMP stimulates protein kinase B in a Wortmannin‐insensitive manner
Authors: Sable Carol L , Filippa Nathalie , Hemmings Brian , Van Obberghen Emmanuel
Published: July 2002

Activation of protein kinase B (PKB) by growth factors has been demonstrated to proceed via phosphatidylinositol 3‐kinase (PI3‐kinase). Here, we show that agents which raise intracellular cAMP can also stimulate PKB. However, this effect is not sensitive to wortmannin, indicating that it is PI3‐kinase independent. This activation does not appear to result from direct phosphorylation by protein kinase A (PKA) since GST–PKB is not an effective PKA substrate. In addition, the activation pathway of PKB by cAMP seems to be linked to that of growth factors, albeit downstream of PI3‐kinase. Evidence for this is that a constitutive active PKB, T308D, S473D, containing activating mutations in the serine and threonine residues which are phosphorylated subsequent to PI3‐kinase activation, cannot be further stimulated by cAMP elevations. Hence, these data suggest that, in addition to growth factors, cAMP can also lead to activation of PKB. This cAMP stimulatory action appears to require phosphorylation of T308 and S473, and hence would indicate that cAMP modulates the phosphorylation event of these PKB regulatory sites.
Comparison of PMM1 with the phosphomannomutases expressed in rat liver and in human cells
Authors: Pirard Michel , Collet Jean-François , Matthijs Gert , Van Schaftingen Emile
Published: July 2002

Carbohydrate‐deficient glycoprotein syndrome type I (CDGI) is most often due to phosphomannomutase deficiency; paradoxically, the human phosphomannomutase gene PMM1 is located on chromosome 22, whereas the CDGI locus is on chromosome 16. We show that phosphomannomutases present in rat or human liver share with homogeneous recombinant PMM1 several kinetic properties and the ability to form an alkali‐ and NH2OH‐sensitive phosphoenzyme with a subunit mass of ≈30 000 M
r. However, they have a higher affinity for the activator mannose‐1,6‐bisphosphate than PMM1 and are not recognized by anti‐PMM1 antibodies, indicating that they represent a related but different isozyme. Phosphomannomutases belong to a novel mutase family in which the active residue is a phosphoaspartyl or a phosphoglutamyl.
Human histidine‐rich glycoprotein expressed in SF9 insect cells inhibits apatite formation
Authors: Schinke Thorsten , Koide Takehiko , Jahnen-Dechent Willi
Published: July 2002

Histidine‐rich glycoprotein (HRG) is structurally related to the α2‐HS glycoprotein/fetuin family of mammalian plasma proteins; both belong to the cystatin superfamily of proteins. We expressed recombinant human HRG and α2‐HS in Sf9 insect cells for functional analysis. Recombinant HRG bound heparin and fibrinogen while α2‐HS did not. Both proteins inhibited the formation of apatite, recombinant HRG (IC50 ∼1 μM) with 2‐fold lower molar activity than α2‐HS (IC50 ∼0.5 μM). The inhibition in vitro of apatite formation suggests a new function for plasma HRG protein, inhibition of phase separation in blood vessels.
Isoform specificity of activators and inhibitors of protein kinase C γ and δ
Authors: Keenan Carolyn , Goode Nigel , Pears Catherine
Published: July 2002

Expression of certain mammalian protein kinase C (PKC) isoforms inhibits the proliferation of Schizosaccharomyces pombe (Goode et al., Mol. Biol. Cell 5 (1994) 907–920). We have taken advantage of this fact to determine the in vivo isoform preference of a number of PKC inhibitors, using a microtitre plate assay which allows rapid screening. This in vivo model has revealed previously unreported preferences; calphostin C is a more efficient inhibitor of the novel PKCδ than chelerythrine chloride whereas the efficiencies are reversed for inhibition of the classical PKCγ. We have also shown that the anti‐leukaemic agent bryostatin 1 inhibits or activates in vivo in an isoform‐specific manner.
Molecular characterization of a new human minisatellite that is able to form single‐stranded loops in vitro and is recognized by nuclear proteins
Authors: Boán Francisco , González Ana Isabel , Rodrı́guez José Manuel , Gómez-Márquez Jaime
Published: July 2002

We report the isolation of a new low polymorphic GC‐rich human minisatellite locus (MsH42) that contains different recombination motifs and is homologous to sequences involved in immunoglobulin class‐switching. Furthermore, we show that MsH42 undergoes slipped‐strand mispairing during PCR indicating its ability to generate single‐stranded loops. Specific DNA‐protein complexes were detected in band‐shifting experiments using nuclear extracts from mouse testes and human NC‐37 cells. The possible implications of this minisatellite in recombination events is discussed.
Specific association of photosystem II and light‐harvesting complex II in partially solubilized photosystem II membranes
Authors: Boekema Egbert J , van Roon Henny , Dekker Jan P
Published: July 2002

In this study, we report the structural characterization of photosystem II complexes obtained from partially solubilized photosystem II membranes. Direct observation by electron microscopy, within a few minutes after a mild disruption of the membranes with the detergent n‐dodecyl‐α,d‐maltoside, revealed the presence of several large supramolecular complexes. Images of these complexes were subjected to multivariate statistical analysis and classification procedures, resolving a new complex consisting of the previously characterized dimeric supercomplex of photosystem II and light‐harvesting complex II [Boekema et al., Proc. Natl. Acad. Sci. USA 92 (1995) 175–179] and two additional, symmetrically organized protein masses each containing a second type of trimeric light‐harvesting II complex. We conclude that large and labile integral membrane proteins, such as photosystem II, can be quickly structurally characterized without extensive purification.
Abscisic acid maintains S‐type anion channel activity in ATP‐depleted Vicia faba guard cells
Authors: Schwarz Martin , Schroeder Julian I
Published: July 2002

The plant hormone abscisic acid (ABA) regulates important developmental and stress responses. Recent data show that ABA activates phosphorylation events, but whether dephosphorylation events are post‐translationally regulated by ABA or whether these are constitutive remains unknown. Slow anion channels in the plasma membrane of guard cells have been proposed to play an important role during ABA‐induced stomatal closing. Anion channels are deactivated by removal of cytosolic ATP. However, when guard cells were treated with ABA and depleted of ATP, anion currents remained active. Subsequent removal of extracellular ABA caused deactivation of currents. Deactivation of currents was reversed by reintroduction of cytosolic MgATP. These data show that anion channels are regulated by ABA even in the absence of cytosolic ATP required for kinase‐induced phosphorylation events and that anion channel activity is maintained by ABA under conditions that favor dephosphorylation‐induced deactivation. Furthermore, channel activation proceeded at high ATP concentrations with nanomolar cytosolic Ca2+ showing a Ca2+‐independent final step in anion channel activation.