bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the structures of the subunits of the cytochrome bc1 complex from beef heart mitochondria
Authors: Link T.A. , Schägger H. , von Jagow G.
Published: July 2002

The interaction of the protein subunits of the bc
1 complex from beef heart is analysed on the basis of protein chemical data and of secondary structure predictions suggesting a large number of amphipathic helices. Electrostatic interactions, i.e. helix‐dipole interactions and ionic bonds, may play a major role in the stabilisation of the arrangement of the subunits within the multi‐protein complex, formation of subcomplexes and maintenance of the steric strain of cytochrome b. A model of the heme‐carrying ‘core’ of cytochrome b, i.e. of helices II‐V, is presented consisting of a twisted ‘4‐α‐helical’ bundle held together by helix‐dipole interactions and stabilised by the interaction with other protein subunits of the bc
1 complex.
Nuclear Overhauser effects in linear peptides A low‐temperature 500 MHz study of Met‐enkephalin
Authors: Motta A. , Tancredi T. , Temussi P.A.
Published: July 2002

Met5‐enkephalin was studied in 1 mM solutions in 2H2O at room temperature and in a cryoprotective mixture (DMSOd6/2H2O, mole fraction of DMSO 0.49) in the temperature range 265–298 K. Small positive effects were observed between the ortho and meta protons of Tyr in aqueous solution at room temperature. Intraresidue effects can be made strong and negative by increasing the viscosity of the medium with a combination of cryoprotective mixtures and low temperatures. The use of mixtures with properties very close to water is very promising for conformational studies of enkephalins and of other small linear peptides.
Nucleotide sequence coding for the human type IV collagen α2 chain cDNA reveals extensive homology with the NC‐1 domain of α1 (IV) but not with the collagenous domain or 3untranslated region
Authors: Hostikka Sirkka Liisa , Kurkinen Markku , Tryggvason Karl
Published: July 2002

We have isolated two overlapping cDNA clones that provide the complete nucleotide sequence coding for the NC‐1 domain and 3′‐untranslated region of the α2 chain of human type IV collagen as well as a sequence encoding 232 residues of the collagenous domain. An extensive homology was observed between the sequences of the NC‐1 domain of the α1(IV) and α2(IV) chains, but considerably less between the sequences encoding collagenous and 3′‐untranslated regions. There were four interruptions in the collagenous sequence studied whereas the comparable region of the α1(IV) chain had only two. A potential oligosaccharide attachment site was found in a 6‐residue long interruption of the collagenous domain but none in the NC‐1 domain.
Selectivity for maltose and maltodextrins of maltoporin, a pore‐forming protein of E. coli outer membrane
Authors: Dargent Bénédicte , Rosenbusch Jürg , Pattus Franc
Published: July 2002

Homogeneous maltoporin (lamB protein), an Escherichia coli outer membrane spanning protein, was incorporated in phospholipid planar bilayers. It generates aqueous channels distinct from those formed by the non‐specific porin (OmpF) or by phosphoporin (phoE protein). The single conductance, 150 pS in 1 M NACl, is much smaller than that of the porins. The channels, which are poorly selective for cations and voltage independent, are specifically inhibited by maltose and maltodextrins. This inhibition, observed in the absence of maltose binding protein, demonstrates that the selectivity of maltoporin for maltose and maltodextrins is an intrinsic property of the protein.
The importance of the negative charge of β‐lactam compounds for the inactivation of the active‐site serine DD‐peptidase of Streptomyces R61
Authors: Varetto Louis , Frère Jean-Marie , Ghuysen Jean-Marie
Published: July 2002

The interaction between the Streptomyces R61 penicillin‐sensitive DD‐peptidase and deacetyl‐cephalosporin C or its lactone derivative has been studied at different pH values. The results show the importance of an enzyme group of pK ≌ 9 which might form an ion pair with the free carboxylate of the former compound. This electrostatic interaction is shown to contribute to the formation of the first, non‐covalent enzyme‐inactivator complex by a factor of at least 50.
Properties of 2′,3′‐dideoxy‐2′,3′‐dehydrothymidine 5′‐triphosphate in terminating DNA synthesis catalyzed by several different DNA polymerases
Authors: Dyatkina Natalia , Minassian Shahnara , Kukhanova Marina , Krayevsky Alexander , von Janta-Lipinsky Martin , Chidgeavadze Zurab , Beabealashvilli Robert
Published: July 2002

2′‐3′‐Dideoxy‐2′,3′‐dehydrothymidine 5′‐triphosphate (dddTTP) shows termination substrate properties in the DNA synthesis catalyzed by E. coli DNA polymerase I KF, rat liver DNA polymerase β, reverse transcriptases of avian myeloblastosis virus and Raus sarcoma virus and calf thymus terminal deoxynucleotidyl transferase. This implies that the mononucleotide residue of dddTTP incorporates into 3′‐termini of newly synthesized DNA chains. However, dddTTP has no influence on the DNA synthesis catalyzed by calf thymus DNA polymerase α. In the case of some DNA polymerases dddTTP was one order of magnitude more effective in comparison with the other known termination substrates.
A photoactivatable synthetic transit peptide labels 30 kDa and 52 kDa polypeptides of the chloroplast inner envelope membrane
Authors: Kaderbhai Mustak A. , Pickering Tracy , Austen Brian M. , Kaderbhai Naheed
Published: July 2002

A 24‐residue transit peptide based on the sequence of a precursor of the small subunit of wheat ribulose‐1,5‐bisphosphate carboxylase (Rubisco) was synthesized. The transit peptide was converted into a radioactive azido derivatised analogue. Photoactivation of the radiolabelled transit peptide analogue with isolated inner and outer membranes of the chloroplast envelope intensely labelled two proteins of 30 kDa and 52 kDa. In the outer membrane only the 52 kDa polypeptide was labelled. These findings are consistent with a recent report on the identification of the 30 kDa receptor protein for protein import in the chloroplast envelope contact zones [(1988) Nature 331, 232–237].
A new photosystem II reaction center component (4.8 kDa protein) encoded by chloroplast genome
Authors: Ikeuchi Masahiko , Inoue Yorinao
Published: July 2002

The photosystem II reaction center complex, so‐called D1‐D2‐cytochrome b‐559 complex, isolated from higher plants contains a new component of about 4.8 kDa [(1988) Plant Cell Physiol. 29, 1233–1239]. The partial amino acid sequence of this component from spinach was determined after release of N‐terminal blockage. The determined sequence matched an open reading frame (ORF36) of the chloroplast genome from tobacco and liverwort, which is located downstream from the psbK gene and forms an operon with psbK. The predicted product consists of 36 amino acid residues and has a single membrane‐spanning segment. High homology between the tobacco and liverwort genes, and its presence in the reaction center complex suggest an important role for this component in the photosystem II complex. Since this gene corresponds to a part of the formerly designated psbI gene, we propose to revise the definition of psbI as the gene encoding the 4.8 kDa reaction center component.
Negative regulation of atrial natriuretic factor receptor coupled membrane guanylate cyclase by phorbol ester Potential protein kinase C regulation of cyclic GMP signal in isolated adrenocortical carcinoma cells of rat
Authors: Jaiswal Rama Kant , Jaiswal Neelam , Sharma Rameshwar K.
Published: July 2002

Rat adrenocortical carcinoma cells possess a high density of atrial natriuretic factor (ANF) receptors which are coupled with membrane guanylate cyclase and corticosterone production. Herein we show that pretreatment of these cells with phorbol 12‐myristate 13‐acetate (PMA), a known activator of protein kinase C, attenuates the ANF‐stimulated cyclic GMP accumulation in a dose‐dependent manner. The half maximum inhibitory concentration of PMA was 10−10 M. When these cells were incubated with PMA in the presence of 1‐(5‐isoquinolinyl‐sulfonyl)‐2‐methyl piperazine, a protein kinase C inhibitor, the PMA‐mediated attenuation of ANF‐stimulated cyclic GMP formation is blocked. These results suggest that protein kinase C negatively regulates the ANF‐receptor coupled membrane guanylate cyclase system in these cells.
The role of long‐chain acyl‐CoA in the damage of oxidative phosphorylation in heart mitochondria
Authors: Borutait≐ V. , Mildaz̆ien≐ V. , Ivanovien≐ L. , Kholodenko B. , Toleikis A. , Pras̆kevic̆ius A.
Published: July 2002

The aim of this investigation was to study the effect of intramitochondrial acyl‐CoA on the respiration of rabbit heart mitochondria over the whole range of stationary respiratory rates between States 4 and 3. The creatine phosphokinase system was used for stabilization of extramitochondrial adenine nucleotide concentration. It was shown that acyl‐CoA depressed respiration more effectively in the intermediate range of respiration between States 4 and 3. The effect of acyl‐CoA was negligible near State 4 and in State 3. These data are in line with our previous results concerning the dependence of the adenine nucleotide translocator control coefficient on the rate of mitochondrial respiration. Thus, our data suggest that long‐chain acyl‐CoA may regulate oxidative phosphorylation in heart mitochondria in vivo.
Ischemia‐induced changes in myocardial paramagnetic metabolites: Implications for intracellular oxy‐radical generation
Authors: Baker John E. , Kalyanaraman B.
Published: July 2002

Evidence to identify the cellular sources of oxy‐radical generation in myocardium has been of an indirect nature. We have used low‐temperature ESR spectroscopy to identify and characterize ischemia‐induced changes in myocardial paramagnetic metabolites. Iron‐sulfur proteins associated with the NADH or succinate dehydrogenases of the mitochondrial electron‐transport chain were progressively reduced with the onset and development of ischemia. This study provides direct evidence for ischemia‐induced changes in an intracellular source of superoxide radical generation that may contribute to oxy‐radical production during reperfusion.
Evidence for a molten globule state as a general intermediate in protein folding
Authors: Ptitsyn O.B. , Pain R.H. , Semisotnov G.V. , Zerovnik E. , Razgulyaev O.I.
Published: July 2002

We propose that the formation of the transient molten globule state occurs early on the pathway of folding of all globular proteins.
Further studies on the human pancreatic binary complexes involving procarboxypeptidase A
Authors: Moulard Maxime , Michon Thierry , Kerfelec Brigitte , Chapus Catherine
Published: July 2002

In contrast to procarboxypeptidase B which has always been reported to be secreted by the pancreas as a monomer, procarboxypeptidase A occurs as a monomer and/or associated to one or two functionally different proteins, depending on the species. Recent studies showed that, in the human pancreatic secretion, procarboxypeptidase A is mainly secreted as a 44 kDa protein involved in at least three different binary complexes. As previously reported, two of these complexes associated procarboxypeptidase A to either a glycosylated truncated protease E or zymogen E. In this paper, we identified proelastase 2 as the partner of procarboxypeptidase A in the third complex, thus reporting for the first time the occurrence of a proelastase 2/procarboxypeptidase A binary complex in vertebrates. Moreover, from N‐terminal sequence analyses, the 44 kDa procarboxypeptidase A involved in these complexes was identified as being of the A1 type. Only one type of procarboxypeptidase B, the B1 type, has been detected in the analyzed pancreatic juices, thus emphasizing the previously observed genetic differences between individuals.
Calmodulin‐dependent multiprotein kinase and protein kinase C phosphorylate the same site on HMG‐CoA reductase as the AMP‐activated protein kinase
Authors: Clarke Paul R. , Grahame Hardie D.
Published: July 2002

Calmodulin‐dependent multiprotein kinase and protein kinase C phosphorylate and inactivate both intact, microsomal HMG‐CoA reductase, and the purified 53 kDa catalytic fragment. Isolation of the single phosphopeptide produced by combined cleavage with cyanogen bromide and Lys‐C proteinase reveals that this is due to phosphorylation of a single serine residue near the C‐terminus, corresponding to serine‐872 in the human enzyme. This is identical with the single serine phosphorylated by the AMP‐activated protein kinase. The nature of the protein kinase responsible for phosphorylation of this site in vivo is discussed.
Does ferredoxin I (Azotobacter) represent a novel class of DNA‐binding proteins that regulate gene expression in response to cellular iron(II)?
Authors: Thomson A.J.
Published: July 2002

Azotobacter vinelandii (Av) and chroococcum (Ac) ferredoxin I contain [3F‐4S]1+/0 and [4Fe‐4S]2+/1+ clusters, when isolated aerobically, which undergo one‐electron redox cycles at potentials of −460±10 mV (vs SHE) at pH 8.3 and −645± 10 mV, respectively. The X‐ray structure of Fd I (Av) reveals that the N‐terminal half of the polypeptide folds as a sandwich of β‐strands which enclose the iron‐sulphur clusters. The C‐terminal sequence contains an amphiphilic α‐helix of four turns which lies on the surface of the β‐barrel. Fd I (Av) controls expression of an unknown protein of Mr
∼ 18 000. Fd I (Ac) will complex iron(II) avidly above pH ∼ 8.0 only when the [3Fe‐4S] cluster is reduced and provided that cellular nucleic acid is bound. Fd I (Ac) rigorously purified from nucleic acid does not undergo iron(II) uptake. These facts, together with recent evidence that the interconversion process [3Fe‐4S]0 + Fe2+ → [4Fe‐4S]2+ in the iron‐responsive element binding protein (IRE‐BP) of eukaryotic cells is controlling protein expression at the level of mRNA (1991, Cell 64, 4771; 1991, Nucleic Acid Res. 19, 1739] leads to the following hypothesis. Fd I is a DNA‐binding protein which interacts by single α‐helix binding in the wide groove of DNA. The binding is regulated by iron(II) levels in the cell. The 7Fe form binds to DNA and represses gene expression. Only the DNA‐bound form of the 7Fe Fd I will take up iron(II), not the form free in solution. Iron(II) becomes bound when the [3Fe‐4S] cluster is reduced. The 8Fe Fd I thus generated no longer binds DNA and the gene is de‐repressed. Sequence comparisons and the crystal structure suggests that the two central turns of the α‐helix are important elements of the DNA‐recognition process and that residues Gln69 and Glu73, which lie on the outer surface of the helix, hydrogen‐bond with specific base pairs.
Identification of a polypeptide bound to the β region of the mouse r protein L32 promoter
Authors: Yoganathan Thillainathan , Sells Bruce H.
Published: July 2002

Studies have been initiated to identify the protein component(s) which interact with the β regulatory region of the mouse ribosomal protein L32 gene promoter. By the combined use of the mobility shift assay and UV cross‐linking, a factor specific for the upstream transcriptional control sequence of the β region of the ribosomal protein L32 promoter has been detected in mouse L1210 nuclear extracts. A mutation (GT→TC at −71 to −70) in this sequence eliminates the binding. β factor is identified as a 55 kDA polypeptide by UV cross‐linking. Addition of excess β element (double‐stranded oligonucleotide) to a cell‐free transcription system reduces transcription of the ribosomal protein L32 gene. Our results provide evidence that the interaction between the β element and the β factor is involved in ribosomal protein L32 transcription.
A study of D52S hen lysozyme‐G1cNAc oligosaccharide complexes by NMR spectroscopy and electrospray mass spectrometry
Authors: Lumb Kevin J. , Aplin Robin T. , Radford Sheena E. , Archer David B. , Jeenes David J. , Lambert Nigel , MacKenzie Donald A. , Dobson Christopher M. , Lowe Gordon
Published: July 2002

The production of a mutant hen lysozyme is described in which Asp‐52, one of the catalytically important residues, is replaced by Ser. The mutant enzyme has very low catalytic activity but NMR studies show that its structure is closely similar to that of the wild‐type protein. NMR experiments also show that well defined complexes are formed with GlcNAc4 and GlcNAc6 bound in the active Site of the mutant enzyme. Then complexes have been examined using electrospray mass spectrometry (ESMS). The most intense peaks arise from the uncomplexed protein indicating that dissociation takes place in the mass spectrometer under the conditions used here. Peaks from minor species corresponding to complexes between the protein and the oligosaccharides are, however, also observed. The possibility that the latter arise from novel covalent enzyme‐saccharide complexes is discussed.
Characterisation of a chloroplast‐encoded sec Y homologue and atpH from a chromophytic alga Evidence for a novel chloroplast genome organisation
Authors: Scaramuzzi Carol D. , Stokes Harold W. , Hiller Roger G.
Published: July 2002

sec Y is a prokaryotic gene that encodes the SecY protein, an integral membrane component of the prokaryotic protein translocation apparatus. A chloroplast‐encoded sec Y homologue has been identified in the unicellular, chromophytic alga, Pavlova lutherii. The gene predicts a protein composed of ten membrane‐spanning regions, that is approximately 25% homologous and 50% similar to bacterial and plastid SecY proteins. The sec Y gene from P. lutherii is independent of the ribosomal protein (rp) gene cluster to which it is closely linked in other organisms. In P. lutherii sec Y is located 5′ to atpI and atpH. Since, in higher plants the atpIHFA gene cluster and the rp gene cluster are separated by approximately 50 kb, we conclude, this indicates a novel chloroplast gene arrangement in P. lutherii.
Comparison of the Ca2+ binding properties of the γ‐carboxyglutamic acid‐containing module of protein Z in the intact protein and in N‐terminal fragments
Authors: Persson Egon , Stenflo Johan
Published: July 2002

Protein Z is a vitamin K‐dependent plasma protein of unknown function. Its modular structure is identical with those of factors VII, IX, X, and protein C. These proteins have an N‐terminal γ‐carboxyglutamic acid (Gla)‐containing module which binds six to ten Ca2+. In factors IX, X, and protein C, the adjacent epidermal growth factor (EGF)‐like module binds one Ca2+ whereas the EGF‐like module in protein Z does not. We have compared the Ca2+ binding properties of a fragment of protein Z comprising the Gla and N‐terminal EGF‐like modules (pZ‐GlaEGFN) with those of intact protein Z and the isolated Gla module by measuring the Ca2+‐induced quenching of the intrinsic protein fluorescence. The similar Ca2+ affinities of pZ‐GlaEGFN and protein Z indicate that pZ‐GlaEGFN has a native conformation and normal Ca2+ binding properties. A comparison of the Ca2+ binding to pZ‐GlaEGFN with those to the corresponding fragments of factors IX, X, and protein C indicate that Ca2+ binding to the N‐terminal EGF‐like modules in the latter proteins does not influence the folding and Ca2+ binding properties of their Gla modules. Furthermore, the Ca2+‐induced fluorescence enhancements of GlaEGF fragments from factors IX, X, and protein C appear to be caused by Ca2+ binding to the site in the EGF‐like modules since it is not observed for pZ‐GlaEGFN.
Placental alkaline phosphatase activity is inversely related to cell growth rate in HeLaS3 cervical cancer cells
Authors: Telfer Joan F. , Green Christopher D.
Published: July 2002

Placental alkaline phosphatase is an inducible enzyme, expressed in HeLaS3 cells, which has been shown to possess protein phosphotyrosine phosphatase activity. Since phosphotyrosine levels are known to increase in actively dividing cells we sought an inverse correlation between PLAP activity and growth rate in HeLaS3 cells. We found that PLAP inducers, Na‐butyrate, dexamethasone, bromodeoxyuridine and dibutyryl cAMP caused a dose‐dependent reduction in growth rate. Mimosine, an agent that blocks the cell cycle in Gl, caused an increase in PLAP activity whilst the mitogen EGF caused a corresponding decrease in PLAP activity. PLAP activity may therefore be related to cell proliferation rate.
NMR determination of the secondary structure and the three‐dimensional polypeptide backbone fold of the human sterol carrier protein 2
Authors: Szyperski T. , Scheek S. , Johansson J. , Assmann G. , Seedorf U. , Wüthrich K.
Published: July 2002

Nuclear magnetic resonance (NMR) spectroscopy was used to determine the secondary structure and the three‐dimensional polypeptide backbone fold of the human sterol carrier protein 2 (hSCP2), which is a basic protein with 123 residues believed to participate in the intracellular transport of cholesterol and various other lipids. Sequence‐specific assignments were obtained for nearly all backbone 1H and 15N resonances, as well as for about two‐thirds of the side‐chain 1H resonances, using uniform15N‐labeling of the protein combined with homonuclear two‐dimensional1H NMR and three‐dimensional 15N‐correlated 1H NMR. Three α‐helices comprising the polypeptide segments of residues 9–22, 25–30 and 78–84 were identified by sequential and medium‐range nuclear Overhauser effects (NOE). The analysis of long‐range backbone‐backbone NOEs showed that hSCP2 further contains a five‐stranded β‐sheet including the residues 33‐41, 47‐54, 60‐62, 71‐76 and 100‐102, which is a central feature of the molecular architecture. The first three strands are arranged in an antiparallel fashion, the polypeptide chain then crosses over this three‐stranded sheet in a right‐handed sense so that the fourth strand is added parallel to the first one. The fifth strand runs antiparallel to the fourth one, so that the overall topology is +1, +1, −3x, −1. The three‐dimensional arrangement of the β‐sheet and the first two helices was determined using an input of 625 NOE upper distance constraints and 95 scalar coupling constants for a preliminary structure calculation with the distance geometry program DIANA.
Electron transport components of the parasitic protozoon Giardia lamblia
Authors: Ellis Jayne E. , Williams Ruth , Cole Deborah , Cammack Richard , Lloyd David
Published: July 2002

The energy metabolism of the intestinal parasite, Giardia lamblia, involves the iron‐sulphur protein, pyruvate:ferredoxin oxidoreductase. Cell fractionation studies showed that this enzyme is associated with the membranes. NADH and NADPH dehydrogenases were found in both the membrane and cytosolic fractions. EPR spectroscopic studies showed the presence of iron‐sulphur clusters in the membrane fraction and in the cytosolic fraction, non‐sedimentable at 6 × 106
g · min. An acidic, soluble protein fraction was separated from the cytosol. It had an EPR spectrum in the reduced state, characteristic of the 2[4Fe‐4S] type of ferredoxin, with g‐factors at 2.04, 1.93 and l .89. and the midpoint redox potential was estimated to be −360 mV. This species is probably a ferredoxin, like those of anaerobic bacteria such as Closlridium and Desiilfovihrio spp. and also that of Entamoeba histolytica. The protein was readily and irreversibly oxidized to give [3Fe 4S] clusters.
Binuclear centre structure of terminal protonmotive oxidases
Authors: Brown Simon , Moody A.John , Mitchell Roy , Rich Peter R.
Published: July 2002

The recent proliferation of data obtained from mutant forms of cytochrome oxidase and analogous enzymes has necessitated a re‐examination of existing structural models. A new model is proposed, consistent with these data, which brings several protonatable residues (Y244, D298, D300, T309, T316, K319, T326) into the vicinity of the binuclear centre, suggestive of a proton‐transferring function. In addition, we also consider those residues which may participate in electron transport between CuA and haem a. We suggest several potential lines of investigation.
Post‐translational processing and Thr‐206 are required for glycosylasparaginase activity
Authors: Fisher Krishna J. , Klein Melissa , Park Hyejeong , Vettese Michelle B. , Aronson Nathan N.
Published: July 2002

Lysosomal glycosylasparaginase is encoded as a 36.5 kDa polypeptide that is post‐translationally processed to subunits of 19.5 kDa (heavy) and 15 kDa (light). Recombinant glycosylasparaginase has been expressed in Spodoptera frugiperda insect cells enabling the precursor and processed forms to be isolated and their catalytic potential determined. Only the subunit conformation was functional indicating glyeosylasparaginase is encoded as an inactive zymogen. The newly created amino terminal residue of the light subunit following maturation, Thr‐206, is believed to be involved in the catalytic mechanism [1992, J. Biol. Chem. 267,6855‐6858]. Here we have constructed two amino acid substitution mutants replacing Thr‐206 with Ala‐206 or Ser‐206 and demonstrate that both destroy enzyme activity.
The nature of the CuAcenter in cytochrome c oxidase
Authors: Malmström Bo G. , Aasa Roland
Published: July 2002

The merits of the suggestion that cuA in cytochrome oxidase is a mixed‐valence binuclear site is reviewed on the basis of recent analytical and spectroscopic studies. First an alternative mononuclear model is presented. Metal analyses indicate that homogeneous oxidase preparations with high activity contain 3Cu/2Fe. Multifrequency EPR measurements demonstrate a close similarity with a copper site in nitrous oxide reductase, and this is also supported by optical and MCD spectra. Strong evidence for a binuclear site is provided by a 7‐line hyperfine structure in the EPR spectra of both enzymes. A binuclear model consistent with amino acid sequence data can be formulated.
Chaperonin GroE and ADP facilitate the folding of various proteins and protect against heat inactivation
Authors: Kawata Yasushi , Nosaka Koji , Hongo Kunihiro , Mizobata Tomohiro , Nagai Jun
Published: July 2002

In the presence of ADP, the molecular chaperones GroEL and GroES from Escherichia coli not only facilitated the refolding of various proteins, but also prevented their irreversible heat inactivation in vitro. Without nucleotides the refolding reactions were arrested by GroEL. Addition of GroES and ADP to the reaction mixture initiated at the refolding reactions and the enzyme activities were regained efficiently. The presence of GroE(GroEL and GroES) and ADP also protected against heat inactivation of native enzymes at various temperatures. These findings suggest that in the presence of GroES, nucleotide binding is an important event in the mechanism of GroEL‐facilitated protein folding.
Tyrosine‐rich acidic matrix protein (TRAMP) is a tyrosine‐sulphated and widely distributed protein of the extracellular matrix
Authors: Forbes Euan G. , Cronshaw Andrew D. , MacBeath Jonathan R.E. , Hulmes David J.S.
Published: July 2002

Tyrosine‐rich acidic matrix protein (TRAMP; 22 kDa extracellular matrix protein; dermatopontin) is a protein that co‐purifies with lysyl oxidase and with dermatan sulphate proteoglycans, with possible functions in cell—matrix interactions and matrix assembly. Using a rabbit polyclonal antiserum raised against porcine TRAMP, which cross‐reacts with both the human and murine forms of the protein, we show by immunoblotting that TRAMP has a widespread tissue distribution, including skin, skeletal muscle, heart, lung, kidney, cartilage and bone. In cultures of human skin fibroblasts, TRAMP incorporates both [35S]sulphate and [3H]tyrosine and is secreted into the medium, as shown by immunoprecipitation. Amino acid analysis of immunoprecipitated TRAMP demonstrates that many of the tyrosine residues in TRAMP are sulphated.
Predicted secondary structure of the 20 S proteasome and model structure of the putative peptide channel
Authors: Lupas Andrei , Koster Abraham J. , Walz Jochen , Baumeister Wolfgang
Published: July 2002

Secondary structure prediction has made great progress in recent years due to the incorporation of evolutionary information, and may be close to a point where (in combination with biochemical and low‐resolution structural data) it can guide the modelling of tertiary structure in cases where no model building is possible by homology. Towards this goal it is important to gather information on the performance of prediction methods in advance of the publication of new structures. In anticipation of the soon‐to‐be‐released structure of the 20 S proteasome from Thermoplasma acidophilum, we have applied several widely used secondary structure prediction methods to proteasome sequences and have attempted to model the putative channel in the outer proteasome rings (α‐rings) based on the obtained predictions.
RNA/DNA hybrid duplexes with identical nearest‐neighbor base‐pairs have identical stability
Authors: Sugimoto Naoki , Katoh Misa , Nakano Shu-ichi , Ohmichi Tatsuo , Sasaki Muneo
Published: July 2002

Energetic behaviors of eight pairs of RNA/DNA hybrid duplexes with identical nearest neighbors have been investigated by UV melting analysis. In the pairs with identical nearest‐neighbor pairs, the melting curve traces at the same strand concentration were very similar. The average difference in stabilization energy of these pairs was 4%, which was about expected within experimental error. These results indicate that the nearest‐neighbor model is valid for predicting the stability of RNA/DNA hybrid duplexes as well as RNA/RNA and DNA/DNA duplexes.
Molecular cloning and expression of subunit 12: a non‐MCP and non‐ATPase subunit of the 26 S protease
Authors: Dubiel Wolfgang , Ferrell Katherine , Dumdey Renate , Standera Sybille , Prehn Siegfried , Rechsteiner Martin
Published: July 2002

A cDNA encoding subunit 12 (S12) of human erythrocyte 26 S protease has been isolated, sequenced and expressed. The cDNA contains an open reading frame that encodes a 36.6 kDA protein 96% identical to mouse Mov‐34 and 67% identical to its Drosophila melanogaster homolog. Based on the high degree of sequence identity between human S12, mouse and Drosophila Mov‐34 proteins, we conclude that the Mov‐34 gene product is a component of the 26 S protease. Antibodies produced against two S12 fragments, Met1‐Tyr95 (S12f95) and Met1‐Leu205 (S12f205), react with S12 transferred to nitrocellulose from SDS‐PAGE. In contrast, after transfer from native gels, the epitope(s) recognized by anti‐S12f205 is exposed in the regulatory complex but appears to be masked when the regulatory complex associates with the multicatalytic protease.
Hormone profile during the menstrual cycle at high altitude
Authors: Escudero F. , Gonzales G.F. , Góñez C.
Published: July 2002

Objectives: To determine the hormone profile during the menstrual cycle in women at sea level and at high altitude, and whether the time between the gonadotropin peak and ovulation is different at high altitude from that at sea level. Methods: The study was carried out in Lima (150 m) and Cerro de Pasco (4340 m), Peru. The hormone profile of 10 adult regularly menstruating women at sea level and 10 women at high altitude was assessed. Ovulation was identified by vaginal ultrasonography. Results: The pre‐ovulatory follicle diameter was lower (P < 0.001) at high altitude than at sea level. Ovulation after LH peak occurred earlier at high altitude than at sea level. Serum FSH levels were higher at late luteal phase and early follicular phase at high altitude than at sea level (P < 0.05). The serum LH and FSH peaks were similar in women at sea level and at high altitude. During the early follicular phase serum estradiol levels were significantly higher at high altitude than at sea level (P < 0.05). During the late follicular phase the production of estradiol was higher at sea level than at high altitude (P < 0.05). The peak of serum estradiol was at day −1 in Lima and in day 0 at high altitude. At ovulation, the serum estradiol levels in women at sea level were 55.1% of the peak, but remained at high levels (80% of the peak) in women at high altitude (P < 0.05). The second increase of serum estradiol occurred earlier at sea level than at high altitude. From days +12 to +15, there was a significant decline in serum estradiol levels in women at sea level (P < 0.05) but not in those from high altitude (P > 0.05). Serum progesterone levels at days +5, and +8 to +12 were significantly higher at sea level than at high altitude. Conclusion: Our data suggest that hormone profile during menstrual cycle is different at high altitude than at sea level, probably as an effect of low barometric pressure.
Glial‐like cells of the rat pituitary intermediate lobe change morphology and shift from vimentin to GFAP expression during development
Authors: Gary Keith A. , Sands Scott A. , Chronwall Bibie M.
Published: July 2002

This study demonstrated morphological changes in glial‐like cells of the rat pituitary intermediate lobe during early postnatal development, and a subsequent shift in protein expression from vimentin to GFAP. Vimentin immunoreactivity was detected in the lobe at embryo day 14 and was localized in radially‐oriented, bipolar cells whose processes spanned the thickness of the intermediate lobe. At electron microscopical resolution, processes contained intermediate filaments, cell nuclei were indented while secretory vesicles characteristic of the endocrine cells were not found. Vimentin immunoreactive intensity began to decrease at postnatal day 5. By postnatal day 7, vimentin‐positive, setellate cells were observed, with few radial processes found by day 10. The intensity of vimentin immunoreactivity decrease through day 25. Within the lobe parenchyma, vimentin was localized in glial‐like cells since double‐label immunohistochemistry revealed no colocalization of β‐endorphin and vimentin, or fibronectin and vimentin. Dopamine‐containing axons were in close apposition to vimentin‐positive processes. GFAP immunoreactivity first appeared on postnatal day 20 and, by day 25, stellate cell bodies with three to six extended processes were evident. Cells were primarily distributed in the caudal third of the lobe. The characteristic adult pattern of cell clusters in latero‐dorsal and ventral portions of the lobe was fully established by postnatal day 55. The transition from vimentin to GFAP expression and concurrent morphological changes resemble those described for radial glia during cerebral cortical development.
The rehabilitation of schizophrenic patients in Austria
Authors: Berner P
Published: July 2002

The foundation of a rehabilitation center for schizophrenics annexed to the Psychiatric University Clinic of Vienna in 1963 initiated a systematic readaptation strategy for these patients all over Austria. The main task of these rehabilitation units is to evaluate the working and social capacities after acute illness episodes, to motivate the patient to accept professional activities and living conditions adapted to their health state through reinforcement of special competences, to replace inadequate attribution and coping styles by appropriate behavior patterns and to enhance compliance for necessary drug treatment. During the rehabilitation period the families are systematically prepared to live with the patients. In accordance with the obtained level of rehabilitation the patients are then either reintegrated into their family and/or normal professional conditions or placed in special settings (homes, sheltered workshops). A long-term followup is assured.
Effect of DNase on the activity of neutrophil elastase, cathepsin G and proteinase 3 in the presence of DNA
Authors: Duranton Jérôme , Belorgey Didier , Carrère Jacqueline , Donato Lionel , Moritz Thomas , Bieth Joseph G.
Published: July 2002

It has been shown previously that DNA binds and inhibits neutrophil elastase (NE). Here we demonstrate that DNA has a better affinity for neutrophil cathepsin G (cat G) than for NE and is a better inhibitor of cat G than of NE. DNase‐generated <0.5 kb DNA fragments inhibit NE and cat G as potently as full length DNA. This rationalises our observation that administration of DNase to cystic fibrosis patients does not enhance the NE and cat G activity of their lung secretions. Neutrophil proteinase 3 is not inhibited by DNA and might thus be the most harmful proteinase in inflammatory lung diseases.
Nascent Lep inserts into the Escherichia coli inner membrane in the vicinity of YidC, SecY and SecA
Authors: Houben Edith N.G. , Scotti Pier A. , Valent Quido A. , Brunner Josef , de Gier Jan-Willem L. , Oudega Bauke , Luirink Joen
Published: July 2002

Targeting and assembly of the Escherichia coli inner membrane protein leader peptidase (Lep) was studied using a homologous in vitro targeting/translocation assay. Assembly of full‐length Lep was efficient in the co‐translational presence of membrane vesicles and hardly occurred when membranes were added post‐translationally. This is consistent with the signal recognition particle‐dependent targeting of Lep. Crosslinking experiments showed that the hydrophilic region P1 of nascent membrane‐inserted Lep 100‐mer was in the vicinity of SecA and SecY, whereas the first transmembrane domain H1 was in the vicinity of YidC. These results suggested that YidC, together with the Sec translocase, functions in the assembly of Lep. YidC might be a more generic component in the assembly of inner membrane proteins.
ADAMTS‐1 cleaves a cartilage proteoglycan, aggrecan
Authors: Kuno Kouji , Okada Yasunori , Kawashima Hiroto , Nakamura Hiroyuki , Miyasaka Masayuki , Ohno Hiroshi , Matsushima Kouji
Published: July 2002

A disintegrin‐like and metalloproteinase with thrombospondin type I motifs‐1 (ADAMTS‐1) is an extracellular matrix‐anchored metalloproteinase. In this study we have demonstrated that ADAMTS‐1 is able to cleave a major cartilage proteoglycan, aggrecan. N‐terminal sequencing analysis of the cleavage product revealed that ADAMTS‐1 cleaves the Glu1871–Leu1872 bond within the chondroitin sulfate attachment domain of aggrecan. In addition, deletional analysis demonstrated that the C‐terminal spacer region of ADAMTS‐1 is necessary to degrade aggrecan. These results suggest that ADAMTS‐1 may be involved in the turnover of aggrecan in vivo.
Characterization of S818L mutation in HERG C‐terminus in LQT2
Authors: Nakajima Tadashi , Kurabayashi Masahiko , Ohyama Yoshio , Kaneko Yoshiaki , Furukawa Tetsushi , Itoh Toshio , Taniguchi Yasuhiro , Tanaka Toshihiro , Nakamura Yusuke , Hiraoka Masayasu , Nagai Ryozo
Published: July 2002

We examined the mechanism(s) for HERG channel dysfunction in an S818L mutation in the HERG C‐terminus using the heterologous expression system in Xenopus oocytes. Injection of S818L cRNA alone did not produce expressed currents. Coinjection of an equal amount of S818L cRNA with wild‐type (WT) cRNA into oocytes did not exhibit apparent dominant‐negative suppression. However, coinjection of excess amounts of S818L cRNAs with WT cRNA into oocytes decreased HERG current amplitudes and shifted the voltage dependence of activation to negative potentials, accelerated its activation and deactivation. The data suggest that S818L alone cannot form functional channels, whereas S818L subunits can, at least in part, coassemble with WT subunits to form heterotetrameric functional channels, and imply that the HERG C‐terminus may contain a domain involving the activation–deactivation process of the channel. These findings may provide new insights into the structure–function relationships of the HERG C‐terminus.
Nuclear and mitochondrial apoptotic pathways of p53
Authors: Moll Ute M , Zaika Alex
Published: July 2002

In contrast to p53‐mediated cell cycle arrest, the mechanisms of p53‐mediated apoptosis in response to cellular stresses such as DNA damage, hypoxia and oncogenic signals still remain poorly understood. Elucidating these pathways is all the more pressing since there is good evidence that the activation of apoptosis rather than cell cycle arrest is crucial in p53 tumor suppression. Moreover, the therapeutic interest in p53 as the molecular target of anticancer intervention rests mainly on its powerful apoptotic capability. This puzzling elusiveness suggests that p53 not only engages a plethora of downstream pathways but itself might possess a biochemical flexibility that goes beyond its role as a mere transcription factor. Recent evidence of a direct pro‐apoptotic role of p53 protein at mitochondria suggests a synergistic effect with its transcriptional activation function and brings an unexpected new level of complexity into p53 apoptotic pathways.
Complementation of bacterial SecE by a chloroplastic homologue
Authors: Fröderberg Linda , Röhl Thomas , van Wijk Klaas-Jan , de Gier Jan-Willem L.
Published: July 2002

The SecE protein is an essential component of the SecAYE‐translocase, which mediates protein translocation across the cytoplasmic membrane in bacteria. In the thylakoid membranes of chloroplasts, a protein homologous to SecE, chloroplastic (cp) SecE, has been identified. However, the functional role of cpSecE has not been established experimentally. In this report we show that cpSecE in cells depleted for bacterial SecE (i) supports growth, (ii) stabilizes, just like bacterial SecE, the Sec‐translocase core component SecY, and (iii) supports Sec‐dependent protein translocation. This indicates that cpSecE can functionally replace bacterial SecE in vivo, and strongly suggests that the thylakoid membrane contains a SecAYE‐like translocase with functional and structural similarities to the bacterial complex. This study further underscores the evolutionary link between chloroplasts and bacteria.
Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via a mevalonate‐independent pathway
Authors: Lichtenthaler Hartmut K , Schwender Jörg , Disch Andrea , Rohmer Michel
Published: July 2002

Isopentenyl diphosphate (IPP) is the biological C5 precursor of isoprenoids. By labeling experiments using [1‐13C]glucose, higher plants were shown to possess two distinct biosynthetic routes for IPP biosynthesis: while the cytoplasmic sterols were formed via the acetate/mevalonate pathway, the chloroplast‐bound isoprenoids (β‐carotene, lutein, prenyl chains of chlorophylls and plastoquinone‐9) were synthesized via a novel IPP biosynthesis pathway (glyceraldehyde phosphate/pyruvate pathway) which was first found in eubacteria and a green alga. The dichotomy in isoprenoid biosynthesis in higher plants allows a reasonable interpretation of previous odd and inconclusive results concerning the biosynthesis of chloroplast isoprenoids, which so far had mainly been interpreted in the frame of models using compartmentation of the mevalonate pathway.
Functional expression of the rat liver canalicular isoform of the multidrug resistance‐associated protein
Authors: Madon Jerzy , Eckhardt Uta , Gerloff Thomas , Stieger Bruno , Meier Peter J
Published: July 2002

The rat hepatocanalicular isoform (called mrp2) of the human multidrug resistance‐associated protein (MRP) has been cloned and transiently expressed in COS‐7 cells and in Xenopus laevis oocytes. In both systems mrp2 expression induced a markedly increased efflux of intracellularly formed [14C]2,4‐dinitrophenyl‐S‐glutathione. Injection of mrp2 cRNA into oocytes also stimulated efflux of [3H(N)]leukotriene C4. Furthermore, mrp2 mRNA was markedly decreased in the liver of the transport mutant TR− rat, which has a congenital defect in the biliary excretion of glutathione‐S conjugates and of other divalent organic anions. The study provides a direct demonstration of mrp2‐mediated transport function and supports the concept that mrp2 represents the canalicular multispecific organic anion transporter (cMOAT) of mammalian liver. © 1997 Federation of European Biochemical Societies.
Molecular organisation of the ice nucleation protein InaV from Pseudomonas syringae
Authors: Schmid Daniel , Pridmore David , Capitani Guido , Battistutta Roberto , Neeser Jean-Richard , Jann Alfred
Published: July 2002

A new ice nucleation gene from Pseudomonas syringae was isolated and overexpressed as a fully active protein in Escherichia coli in order to gain experimental data about the structure of ice nucleation proteins. No evidence of a signal sequence or secondary glycosylation was found. Differences in the extent of aggregation were shown to modulate the ice nucleation activity. The circular dichroism spectrum of the purified protein indicated the presence of β‐sheet structure. This finding supports a recently proposed hypothetical model for the structure of ice nucleation proteins, which provides a plausible explanation for their aggregation tendency.
A sulfatide receptor distinct from L‐selectin is involved in lymphocyte activation
Authors: Ding Ziqiang , Kawashima Hiroto , Suzuki Yasuo , Suzuki Takashi , Ward Peter A , Miyasaka Masayuki
Published: July 2002

Sulfatide induces leukocyte activation, which is thought to be mediated via L‐selectin. Here we demonstrate that lymphocytes express a receptor for sulfatide distinct from L‐selectin and that this receptor is involved in sulfatide‐induced cell activation. While cell surface L‐selectin expression was abolished by phorbol 12‐myristate 13‐acetate (PMA), lymphocytes retained the ability to bind sulfatide in liquid phase as well as in immobilized solid phase. The novel sulfatide receptor obtained from PMA‐treated lymphocytes showed a molecular size of 65 kDa. Stimulation through this receptor triggered cytosolic free Ca2+ elevation and intercellular aggregation.
The mode of action of peptidyl prolyl cis/trans isomerases in vivo: binding vs. catalysis
Authors: Fischer Gunter , Tradler Thomas , Zarnt Toralf
Published: July 2002

Polypeptides often display proline‐mediated conformational substates that are prone to isomer‐specific recognition and function. Both possibilities can be of biological significance. Distinct families of peptidyl prolyl cis/trans isomerases (PPIases) evolved proved to be highly specific for proline moieties arranged in a special context of subsites. Structural and chemical features of molecules specifically bound to the active site of PPIases served to improve catalysis of prolyl isomerization rather than ground state binding. For example, results inferred from receptor Ser/Thr or Tyr phosphorylation in the presence of site‐directed FKBP12 mutant proteins provided evidence for the crucial role of the enzymatic activity in down‐regulating function of FKBP12.
Maize polyamine oxidase: primary structure from protein and cDNA sequencing
Authors: Tavladoraki Paraskevi , Schininà M.Eugenia , Cecconi Francesco , Di Agostino Silvia , Manera Francesco , Rea Giuseppina , Mariottini Paolo , Federico Rodolfo , Angelini Riccardo
Published: July 2002

The first complete amino acid sequence of a flavin‐containing polyamine oxidase was solved by a combined approach of nucleotide and peptide sequence analysis. A cDNA of 1737 bp, isolated from maize seedlings by reverse transcription‐polymerase chain reaction and rapid amplification of cDNA ends strategies, was cloned and its sequence determined. This cDNA contains information for a polypeptide chain of 500 amino acids. Its amino‐terminal sequence shows the typical features of secretion signal peptides. The primary structure of the mature protein was independently confirmed by extensive amino acid sequencing. Structural relationships with flavin‐containing monoamine oxidases are also discussed.
Localization of fractalkine and CX3CR1 mRNAs in rat brain: does fractalkine play a role in signaling from neuron to microglia?
Authors: Nishiyori Atsushi , Minami Masabumi , Ohtani Yoshikazu , Takami Shinya , Yamamoto Junki , Kawaguchi Nami , Kume Toshiaki , Akaike Akinori , Satoh Masamichi
Published: July 2002

Localization of the mRNAs for fractalkine, a CX3C chemokine, and for its receptor CX3CR1 was investigated in the rat brain. In situ hybridization study revealed that fractalkine mRNA was dominantly expressed in neuronal cells particularly in the olfactory bulb, cerebral cortex, hippocampus, caudate putamen and nucleus accumbens. In vitro study using enriched neuronal or glial culture supported the dominant expression of fractalkine mRNA in neurons. On the other hand, CX3CR1 mRNA was dominantly expressed in glial cells throughout the whole brain. The in vitro study suggested the cells expressing CX3CR1 mRNA are microglia, not astrocytes or neurons. Fractalkine appears to function as a signal molecule from neuron to microglia.