bluuebunny/crossref_metadata_2025 huggingface dataset:
Monitoring Fusarium toxins from barley to malt: Targeted inoculation with Fusarium culmorum
Authors: Biehl Eva Maria , Schneidemann-Bostelmann Sarah , Hoheneder Felix , Asam Stefan , Hückelhoven Ralph , Rychlik Michael
Published: December 2024

Molds of the genus Fusarium infect nearly all types of grain, causing significant yield and quality losses. Many species of this genus produce mycotoxins, which pose significant risks to human and animal health. In beer production, the complex interaction between primary fungal metabolites and secondarily modified mycotoxins in barley, malt, and beer complicates the situation, highlighting the need for effective analytical methods to quickly and accurately monitor these toxins. We developed and validated a liquid chromatography-tandem mass spectrometry (LC–MS/MS) method to simultaneously analyze 14 Fusarium toxins, including modified forms (deoxynivalenol (DON), DON-3-glucoside, 3-acetyl-DON, 15-acetyl-DON, nivalenol, fusarenone X, HT-2 toxin, T-2 toxin, the enniatins A, A1, B, B1, beauvericin, and zearalenone) in barley and throughout the malting process. Stable isotope dilution assays (SIDAs) and matrix-matched calibration were used for quantification. A micro-malting setup was established to produce Fusarium-contaminated barley malt under reproducible conditions using targeted inoculation with F. culmorum. Mycotoxins were quantified throughout the malting process and compared to the content of fungal DNA. Further, the impact of various malting parameters was investigated, thus revealing that different malting scenarios exhibited different toxin enrichment patterns. We demonstrated that mycotoxin concentration and the ratio of DON to DON-3-glucoside changed throughout the malting processes, depending on fungal spore concentrations, germination temperature, and malting temperature. The study highlights the complexity of mycotoxin dynamics in malt production and the importance of optimized processing conditions to minimize toxin levels in final malt products.
Assessing seismic performance of mid-rise reinforced concrete buildings using fragility curves
Authors: Naiel Ahmed , Hanna Nagy , Lotfy Ibrahim
Published: December 2024

This paper focuses on the seismic fragility assessment of mid-rise reinforced concrete (RC) residential buildings in Cairo, Egypt. Structural safety is assessed by utilizing the damage that could occur to main components, such as columns and shear walls, and consequently, their failure that may lead to progressive (or partial) collapse of structures under extreme dynamic loads. Behaviors of mid-rise reinforced concrete (RC) buildings, which are designed in accordance with the Egyptian standards, are assessed analytically under the action of seismic loads. As such, non-linear time-history analyses (NLTHA), in addition to pushover analyses, were carried out on three (RC) frames with different number of stories varying from six to ten floors. In addition, fragility curves were developed in order to illustrate the probability of exceeding structural damage due to earthquakes as a function of ground motion indices. The lateral response of previously mentioned (RC) frames are evaluated by utilizing more than eighteen hundred NLTHA considering ten different earthquake records with fourteen peak ground acceleration in addition to the Egyptian response spectrum analysis. The output of this research includes roof drift and base shear histories, which are converted to damage in beams and columns in accordance with FEMA-356 guidelines. Based on statistics and probability distribution functions for the obtained results, fragility curves were then developed, which can be used as an effective tool for planning and implementing retrofit schemes, modifications to codified seismic provisions, seismic mitigation strategies, and for insurance-related applications.
To use or not to use: exploring the ethical implications of using generative AI in academic writing
Authors: Rentier Eline S.
Published: December 2024

The rapid emergence of open-access generative artificial intelligence (GenAI) has sparked a heated debate with polarising views on whether or not it is ethical to use GenAI for academic writing. In this commentary, I explore the ethical implications of using GenAI in academic writing. I provide an overview of how GenAI works as a probabilistic model and what the limitations are, such as generating incorrect or entirely made-up information. I raise concerns about bias, misinformation, plagiarism, inequity of access and monetisation of knowledge and discuss how this implicates ethical values regarding plagiarism and research integrity. Despite these issues, I also acknowledge the potential of GenAI to advance academic writing by aiding in tasks like structuring text and summarising large bodies of information. I emphasise the need to be transparent about the use of GenAI and call for an open discussion on this contentious, yet important, topic.
Complexes of cytochrome P450 with metyrapone
Authors: Mitani Fumiko , Shephard Elizabeth A. , Phillips Ian R. , Rabin Brian R.
Published: July 2002

Phenobarbital‐inducible cytochrome P450 variants have a higher affinity (app. K
s = 0.6 μM) for metyrapone than do non‐phenobarbital‐inducible variants (app. K
s = 80 μM). The difference in affinity has enabled us to quantitate, by metyrapone‐induced difference spectral analysis, phenobarbital‐inducible cytochromes P450 in liver microsomal membranes. The result obtained by this simple, rapid technique compare well with those found by more sophisticated techniques such as radioimmunoassay and immunoprecipitation.
Hydrogen‐bonded conformation of hyaluronate oligosaccharide fragments in aqueous solution
Authors: Oberholtzer J.C. , Englander S.W. , Horwitz A.F.
Published: July 2002

The hydrogen bonding in hyaluronate oligosaccharide fragments was studied in aqueous solution using hydrogen‐tritium exchange techniques. The data reveal an acetamido hydrogen exchange rate that is 5–6‐fold slower than that seen in model compounds. The magnitude of the slowing is interpreted as reflecting the participation of an acetamido hydrogen in a relatively labile intramolecular hydrogen bond.
Pore‐forming activity in the outer membrane of the chloroplast envelope
Authors: Flügge U.Ingo , Benz Roland
Published: July 2002

The permeability properties of the outer membrane of the chloroplast envelope were studied. The exclusion limit for the penetration of molecules through this membrane into the intermembrane space lies between M
r values of about 7000–13 000. This is the largest exclusion limit measured so far for pores of the porin type. In addition, the pore‐forming activity of the outer membrane of the chloroplast envelope was measured by reconstitution experiments. The pore has a single channel conductance of 0.72 nS, which is a linear function of the salt solution in the aqueous phase. The diameter of the chloroplast porin was calculated to be 2.5–3 nm, which is the largest diameter of all known porin pores, e.g., from mitochondria or Gram‐negative bacteria.
Rapid increase of mitochondrial uncoupling protein and its mRNA in stimulated brown adipose tissue
Authors: Ricquier Daniel , Mory Gérard , Bouillaud Frédéric , Thibai Jean , Weissenbach Jean
Published: July 2002

The increase in mitochondrial uncoupling protein in brown adipose tissue during acute stimulation by exposure of animals to cold was examined. Uncoupling protein level increased during the first hours of tissue stimulation. Use of a cDNA probe shows that synthesis of uncoupling protein mRNA was quickly stimulated. Animals treated with propranolol exhibited neither increase in uncoupling protein mRNA nor increase in the protein itself.
A comparison of the primary structures of the two B800‐850‐apoproteins from wild‐type Rhodopseudomonas sphaeroides strain 2.4.1 and a carotenoidless mutant strain R26.1
Authors: Theiler Rolf , Suter Franz , Zuber Herbert , Cogdell Richard J.
Published: July 2002

The α‐ and β‐apoproteins from the B800‐850‐complex of Rhodopseudomonas sphaeroides strain 2.4.1 have been sequenced. These results have been compared with those previously obtained with the analogous antenna apoproteins from the carotenoidless mutant R26.1 [9]. The α‐apoproteins differ at position 24, where a valine residue present in the wild‐type sequence is replaced by a phenylalanine residue in the mutant. The α‐apoproteins differ at the N‐terminus. In the wild‐type β‐apoprotein some chains have methionine at the N‐terminus and some have an N‐terminal threonine, while in the mutant the N‐terminus is threonine.
A structural and dynamic molecular model for the sodium channel of Electrophoruselectricus
Authors: Kosower Edward M.
Published: July 2002

Chemical logic and single group rotation (SGR) theory are applied to the primary structure determined by Noda [(1984) Nature 312, 121‐127] to construct a molecular model of the sodium channel of Electrophorus electricus. Both structural and dynamic aspects of the channel are accounted for, including gating current, sensitivity to changes in membrane potential, channel opening, a binding site for sodium, selectivity for sodium over potassium, capacity for rapid sodium flow, sensitivity to batrachotoxin (or other toxins) and inactivation.
Human brain fibroblast growth factor
Authors: Böhlen Peter , Esch Frederick , Baird Andrew , Jones Kenneth L. , Gospodarowicz Denis
Published: July 2002

Fibroblast growth factor (FGF) has been purified to homogeneity from human brain by a procedure involving salt precipitation, cation‐exchange chromatography, Heparin‐Sepharose affinity chromatography and reverse‐phase HPLC. Isolation was monitored by radioimmunoassay and/or by testing column fractions for their capacity to stimulate the proliferation of vascular endothelial cells in vitro. The amino‐terminal sequence of human brain FGF was, determined as Pro‐Ala‐Leu‐Pro‐Glu‐Asp‐Gly‐Gly‐Ser‐Gly‐Ala‐PhePro‐. This sequence is identical to that of the amino‐terminal region of bovine FGF. Additional evidence, including amino acid composition, Chromatographie retention behavior, and immunoreactivity suggest that the human and bovine mitogens are very similarin structure.
Induction of drug metabolizing enzymes in human liver cell line Hep G2
Authors: Dawson J.R. , Adams D.J. , Wolf C.R.
Published: July 2002

Human cytochrome P‐450, UDP‐glucuronosyltransferase and sulphotransferase activities have been measured in the cell line Hep G2 following treatment of cells with 3‐methylcholanthrene or phenobarbital. 3‐Methylcholanthrene treatment caused a 20–30‐fold increase in the O‐deethylation of 7‐ethoxycoumarin. The glucuronidation and sulphation of the product 7‐hydroxycoumarin were increased 36 and 7 fold, respectively. In comparison, phenobarbital treatment did not increase these activities significantly. However, phenobarbital‐inducible proteins were identified on ‘Western blots’ using antibodies to a rat liver phenobarbital inducible P‐450 form. The molecular masses of the proteins did not coincide with those expected for cytochromes P‐450. However, characteristic of P‐450 forms, the synthesis of these proteins was suppressed by 3‐methylcholanthrene treatment. The Hep G2 cell line represents a potentially useful model for studying the regulation of human P‐450 genes.
The influence of P‐light chain phosphorylation by myosin light chain kinase on the calcium sensitivity of chemically skinned heart fibres
Authors: Morano I. , Hofmann F. , Zimmer M. , Rüegg J.C.
Published: July 2002

Phosphorylation of the P‐light chain of myosin might be involved in the regulation of cardiac contractility. Thus an enhanced phosphorylation level of the P‐light chain catalyzed by Ca2+‐calmodulin‐dependent myosin light chain kinase (MLCK) increased significantly the Ca2+ sensitivity of chemically skinned ventricular fibre bundles of the pig. This effect was reversible. Whereas force development at submaximal Ca2+ concentration (pCa 5.5) increased by ~ 50% in the presence of MLCK, maximum tension achieved at maximum Ca2+‐concentration (pCa 4.3) was not affected.
Effects of GUG and AUG initiation codons on the expression of lacZ in Escherichia coli
Authors: Looman A.C. , van Knippenberg P.H.
Published: July 2002

We have replaced the ribosomal binding site (RBS) of the lacZ gene of E. coli by those of the maturation (A) gene of phage MS2 and that of the tufA gene. Both RBSs contain a GUG initiation codon. The expression with the tufA RBS is at least 25‐fold higher than with the phage RBS. Changing the GUG into AUG results in a 3‐fold increase in expression in both cases. In general, higher expression is accompanied by an increase of lac‐specific mRNA. It is argued that this is a consequence of the more efficient translation of the mRNA.
Domain organization of the terminal parts in the fibrinogen molecule
Authors: Medved' L.V. , Litvinovich S.V. , Privalov P.L.
Published: July 2002

Using limited proteolysis and scanning microcalorimetry it was shown that each terminal part of the fibrinogen molecule is constituted by four co‐operative domains. Among these domains two strongly interacting domains are formed by the C‐terminal part of the β‐chain, while the two other domains are formed by the C‐terminal part of the γ‐chain.
31P‐NMR studies of respiratory regulation in the intact myocardium
Authors: From Arthur H.L. , Petein Marc A. , Michurski Steven P. , Zimmer Stevan D. , Uǧurbil Kâmil
Published: July 2002

The mechanism by which mitochondrial respiration is coupled to ATP consumption in intact tissues is unclear. We determined the relationship between high‐energy phosphate levels and oxygen consumption rate in rat hearts operating over a range of workloads and perfused with different substrates. With pyruvate + glucose perfusion, ADP levels were in general very low, and varied with MVO2 yielding an apparent Km of 25 ± 5 μM, suggesting regulation of oxidative phosphorylation through availability of ADP. In contrast, with glucose perfusion in the presence or absence of insulin, ADP levels, ADP/ATP ratio or the phosphate potential were relatively constant over the workload range examined and generally not correlated with alterations in MVO2; it is suggested that under these conditions, carbon substrate delivery to the mitochondria may control mitochondrial respiration. The common feature of both of the suggested regulatory mechanisms is substrate limitation which, however, is exercised at different metabolic points depending on the carbon substrate available to the myocardium.
The glycine‐rich loop of adenylate kinase forms a giant anion hole
Authors: Dreusicke Dirk , Schulz Georg E.
Published: July 2002

The conformation of the glycine‐rich loop of adenylate kinase is described in detail. It forms a giant anion hole for a sulfate ion, which presumably mimicks a nucleotide phosphoryl group. This loop had been called flexible, because at pH values of 6 or below it is displaced in the crystal. In the region of this loop the adenylate kinases are probably homologous to the p21 proteins. Is is known that a mutation in this loop at residue 12 of p21 causes cell transformation and therefore cancer. Other potentially homologous proteins are indicated.
A positive relationship between protein synthetic rate and intracellular glutamine concentration in perfused rat skeletal muscle
Authors: MacLennan Peter A. , Brown R.A. , Rennie Michael J.
Published: July 2002

During muscle‐protein wasting associated with injury and disease the distribution ratio of free glutamine between muscle and blood falls. In pursuing possible consequences of this, we investigated the relationship between the rate of muscle protein synthesis and intramuscular glutamine concentration, manipulated acutely in the isolated perfused rat hindquarter. Increasing perfusate glutamine from 0.67 to 5.0 mM caused a 200% increase in intracellular glutamine and a 66% increase in protein synthesis in the absence of insulin; in the presence of insulin a 30% increase in intramuscular glutamine was accompanied by an 80% increase in protein synthesis. Analysis of variance of the results confirmed the existence of positive relationships between intramuscular glutamine and protein synthesis in the presence or absence of insulin. Control of the size of the intramuscular free pool of glutamine may be important in determining the muscle protein mass.
Protein kinase C phosphorylates tau and induces its functional alterations
Authors: Hoshi Minako , Nishida Eisuke , Miyata Yoshihiko , Sakai Hikoichi , Miyoshi Tomoko , Ogawara Hiroshi , Akiyama Tetsu
Published: July 2002

We found that tau, one of the major microtubule‐associated proteins, is a good substrate for protein kinase C. The phosphorylation occurred mainly on serine residues and the sites phosphorylated by protein kinase C were largely different from those phosphorylated by cAMP‐dependent protein kinase as analyzed by phosphopeptide mapping. The protein kinase C‐mediated phosphorylation of tau reduced its abilities to promote tubulin polymerization and to cross‐link actin filaments. The reduction in its abilities was in proportion to the number of phosphates incorporated into tau.
Molecular cloning of the myelin specific enzyme 2′,3′‐cyclic‐nucleotide 3′‐phosphohydrolase
Authors: Vogel Ursula S. , Thompson R.J.
Published: July 2002

We describe the isolation of cDNA clones for bovine brain 2′,3′‐cyclic‐nucleotide 3′‐phosphohydrolase (CNPase, EC 3.1.4.37), the third most abundant protein in central nervous system myelin. The cDNA encodes the complete protein (400 amino acids) and hybridizes to a major size species of mRNA in bovine brain tissue, approx. 2.7 kb in size. CNPase mRNA levels do not appear to be affected in quaking dysmyelinating mutant mice. The sequence reveals probable sites for CNPase phosphorylation by cAMP‐dependent protein kinase and a region of homology with haemocyanin.
Identification of protein phosphatase 2A as the major tyrosine hydroxylase phosphatase in adrenal medulla and corpus striatum: evidence from the effects of okadaic acid
Authors: Haavik Jan , Schelling Donald L. , Campbell David G. , Andersson Kristoffer K. , Flatmark Torgeir , Cohen Philip
Published: July 2002

(i) The major sites on bovine adrenal tyrosine hydroxylase (TH) phosphorylated by calmodulin‐dependent multiprotein kinase (CaM‐MPK) and cyclic AMP‐dependent protein kinase were shown to be Ser‐19 and Ser‐40, respectively, while Ser‐40 was also phosphorylated slowly by CAM‐MPK. (ii) Type 2A and type 2C phosphatases accounted for ≈90% and ≈ 10% of TH phosphatase activity, respectively, in extracts of adrenal medulla and corpus striatum assayed at near physiological free Mg2+(1 mM), while type 1 and type 2B phosphatases had negligible activity towards TH. (iii) Incubation of adrenal chromaffin cells with okadaic acid increased TH phosphorylation by 206% and activity by 77%, establishing that type 2A phosphatases play a major role in regulating TH in vivo.
Photocrosslinking demonstrates proximity of a 34 kDa membrane protein to different portions of preprolactin during translocation through the endoplasmic reticulum
Authors: Wiedmann Martin , Goerlich Dirk , Hartmann Enno , Kurzchalia Teymuras V. , Rapoport Tom A.
Published: July 2002

Photocrosslinking has been used to identify integral proteins of the endoplasmic reticulum membrane that are in proximity to nascent preprolactin during in vitro translocation. A photoreactive lysyl derivative was introduced into truncated preprolactin chains comprising 86 or 115 amino acids. Both with the 86mer, containing the reactive group in the signal sequence, and with the 115mer, containing the probe exclusively in the mature portion of the chain, photocrosslinking occurred to a 3̃5 kDa transmembrane glycoprotein, the signal sequence receptor (SSR). SSR is identical with a previously isolated abundant and ubiquitous 34 kDa membrane protein that appears to be essential for protein translocation.
Pharmacological characterization and region‐specific expression in brain of the β2‐ and β3‐subunits of the rat GABAA receptor
Authors: Lolait Stephen J. , O'Carroll Anne-Marie , Kusano Kiyoshi , Mahan Lawrence C.
Published: July 2002

The cDNA for a third β‐subunit of the rat GABAa receptor has been cloned using another β‐subunit, which we had previously cloned [(1989) FEBS Lett. 246, 145‐148], as a probe. The ~ 8‐kb cDNA for this β‐subunit (termed β2) encodes a protein of 474 amino acid residues that shares ~ 80% sequence identity with the rat and bovine β1‐ and β3‐subunits. Coexpression of the cloned β‐subunit cDNA with the α1‐subunit cDNA of the rat GABAa receptor in Xenopus oocytes produced a functional receptor and Cl− channel with pharmacological characteristics of a GABAa receptor. In contrast to interchanging α‐subunits [(1988) Nature 335, 76‐79], exchange of β2‐ or β3‐subunits in α1/β receptor complex did not markedly alter the pharmacological properties of expressed receptors. In situ hybridization histochemistry with synthetic subunit‐specific oligodeoxynucleotide probes revealed a region‐specific expression of α1‐, β2‐ and β3‐subunit mRNAs in the rat central nervous system. These observations provide an additional molecular basis for the functional heterogeneity in the GABAA receptor complex.
Identification of a group of proteins that are strongly up‐regulated in total epidermal keratinocytes from psoriatic skin
Authors: Celis Julio E. , Cruger Dorthe , Kiil Jette , Lauridsen Jette B. , Ratz Gitte , Basse Bodil , Celis Ariana
Published: July 2002

Analysis using two‐dimensional (2D) gel electrophoresis of the [35S]‐methionine‐labelled proteins synthesized by non‐cultured total epidermal keratinocytes obtained from normal and psoriatic skin revealed 6 proteins that are strongly up‐regulated (5 times or more) in psoriatic skin. These proteins are synthesized at albeit lower levels by keratinocytes from normal and normal‐appearing (uninvolved) skin of psoriatic patients, and correspond to isoelectric focusing sample spot numbers 4311 (40.3 kDa), 4003 (12.4 kDa), 5008 (11.9 kDa), 3012 (11.6 kDa), 6016 (11.6 kDa) and 1015 (10;1 kDa) in the normal keratinocyte 2D gel protein database [Celis et al, (1990) Electrophoresis, in press]. These proteins are also detected in the labelling medium indicating that they are at least in part secreted. Given their striking regulatory behavior, these proteins may play a role in the pathogenesis of psoriasis.
Enhanced secretion of endothelin‐1 by elevated glucose levels from cultured bovine aortic endothelial cells
Authors: Yamauchi Teruaki , Ohnaka Keizo , Takayanagi Ryoichi , Umeda Fumio , Nawata Hajime
Published: July 2002

We have investigated the effect of glucose on the release of endothelin‐1‐like immunoreactivity (ET‐l‐LI) from cultured bovine aortic endothelial cells. Elevation of glucose concentrations in cultured media from 5.5 to 11.1 or 22.2 mM significantly stimulated ET‐1‐LI release from cultured endothelial cells. An aldose reductase inhibitor did not affect the high glucose‐induced ET‐1‐LI release, These findings suggest the possibility that hyperglycemia in diabetic patients enhances ET‐1‐LI release at the local site of vascular endothelium. which might be involved in the developments of vascular complications and atherosclerosis.
Desferrioxamine as a lipid chain‐breaking antioxidant in sickle erythrocyte membranes
Authors: Hartley Andrew , Davies Michael , Rice-Evans Catherine
Published: July 2002

The mechanism of action of desferrioxamine in the inhibition of the catalysis of iron‐induced oxidative damage has been ascribed to its ability to chelate available ferric ion (K
b = 1031). However, recent work has proposed that the trihydroxamate moiety of desferrioxamine can also be involved in electron transfer reactions involving the Superoxide radical, peroxidase/hydrogen peroxide mixtures and ferryl myoglobin radicals. In this study we report evidence for the ability of desferrioxamine to inhibit peroxidative damage to pathological membranes with which non‐haem iron is associated through a mechanism of action as a lipid chain breaking antioxidant, independently of its iron chelating properties.
Primary structure of cAMP‐gated channel from bovine olfactory epithelium
Authors: Ludwig Jost , Margalit Talya , Eismann Elisabeth , Lancet Doron , Kaupp U.Benjamin
Published: July 2002

The complete amino‐acid sequence of the bovine olfactory epithelium adenosine 3',5'cyclic monophosphate (cAMP)‐gated channel has been determined by cloning and sequencing its cDNA. It exhibits a high degree of sequence homology with the cGMP‐gated channel of rod photoreceptors, suggesting that cyclic nucleotide‐gated channels fall into a new family of genetically related proteins.
Geometry of binding of the Nα‐tosylated piperidides of m‐amidino‐, p‐amidino‐ and p‐guanidino phenylalanine to thrombin and trypsin
Authors: Turk Dušan , Stürzebecher Jörg , Bode Wolfram
Published: July 2002

The X‐ray crystal structures of the complexes formed with bovine trypsin and the Nα‐tosylated piperidides of m‐amidino‐, p‐amidino‐ and p‐guanidino‐D,L‐phenylalanine (3‐TAPAP, 4‐TAPAP and 4‐TGPAP) were determined with data to 1.8 Å resolution. The L‐stereoisomer of 3‐TAPAP binds as a compact entity into the active site of trypsin, with the amidino and the carbonyl groups of the central amidinophenylalanyl residue hydrogen‐bonded to Gly216 of trypsin. According to modeling and energy minimization, 3‐TAPAP fits perfectly in this conformation to the more restrictive thrombin active site also (Bajusz et al. (1978) Int. J. Pept. Prot. Res. 12, 217‐221); the piperidine moiety extends into the cage‐like S2 subsite of thrombin, but leaves room for additional substituents which might help to improve binding and pharmacological properties. In contrast, 4‐TAPAP and 4‐TGPAP bind only weakly and in an extended conformation to trypsin; their considerably enhanced affinities for thrombin would suggest a more compact binding to thrombin.
Stabilization of clathrin coated vesicles by amantadine, tromantadine and other hydrophobic amines
Authors: Phonphok Yupa , Rosenthal Ken S.
Published: July 2002

Amantadine and related compounds stabilized the structure of purified pig brain clathrin coated vesicles (CCV) at biologically relevant concentrations. Incubation of purified CCV for 30 min at 25°C or 37°C caused the release clathrin, as determined by a centrifugation assay, and a reduction in the number of coated vesicles, by electron microscopy. Amantadine (10 mM), tromantadine (1 mM), amidine D295 (cyclohexylcarboximidamide‐(N‐benzyl)hydrochloride) (10 mM), chloroquin (0.1 mM) and monodansyleadaverine (10 mM) significantly reduced the extent of dissociation.
Small weak acids stimulate proton transfer events in site‐directed mutants of the two ionizable residues, GluL212 and AspL213, in the QB‐binding site of Rhodobacter sphaeroides reaction center
Authors: Takahashi Eiji , Wraight Colin A.
Published: July 2002

Mutations of the two ionizable residues. GluL212 and AspL213, in the secondary quinone (Q#) binding site of reaction centers (RCs) from Rhodobacter sphaeroides cause major dysfunctions in the proton transfer processes leading to the formation of quinol. Mutant RCs with AspL213→ Asn are especially severely blocked, and the rate of the proton‐limited transfer of the second electron is at least IO4 times slower than in the wild‐type. Small, weak acids, such as azide/hydrazoic acid (N3
−/HN3; pϰ ∼ 4.7) accelerated the electron transfer rate in mutant RCs in a pH and concentration‐dependent manner, consistent with their functioning as protein‐penetrating protonophores, delivering protons to the Q# site in a non‐specific, diffusive process. Other small weak acids similarly with efficacies dependent on their size and pϰ values. In terms of the concentration of protonated species, the relative effectiveness was: nitrite > cyanate & sim; formate > azide > > acetate. The behavior of bacterial RCs containing the AspL213→ Asn mutation resembles that of bicarbonate‐depleted photosystem II, and the mutational block is partially alleviated by bicarbonate. The possibility is discussed that bicarbonate acts in PS II as an analogue to the carboxylic acid residues of the bacterial proton conduction pathway.
Hyperreactivity of adenines and conformational flexibility of a translational repression site
Authors: Talbot Simon J. , Medina Gloria , Fishwick Colin W.G. , Haneef I. , Stockley Peter G.
Published: July 2002

We have used a diethylpyrocarbonate (DEPC) modification [(1976) Prog. Nucl. Acids Res. 16, 189–262] to probe the accessibility of adenines essential for coat protein binding in the MS2 translational operator [(1983) Biochemistry 22, 2601–2610, 2610–2615, 4723–4730; (1987) Biochemistry 26, 1563–1568]. The essential adenines are apparently hyperreactive with this reagent relative to other sites within the same molecule. Variation of ionic strength, pH and divalent cation concentrations reveal the existence of two distinct conformers of the RNA operator as judged by DEPC reactivity. We propose that the hyperreactivity observed is due to the participation of neighbouring bases in the DEPC modification reaction and can be used as a novel structural probe.
Dexamethasone induces the expression of the mRNA of lipocortin 1 and 2 and the release of lipocortin 1 and 5 in differentiated, but not undifferentiated U‐937 cells
Authors: Solito Egle , Raugei Giovanni , Melli Marialuisa , Parente Luca
Published: July 2002

The effect of dexamethasone on mRNA and protein synthesis of lipocortins (LCT) 1, 2 and 5 has been investigated in U‐937 cells. A constitutive expression of both mRNAs and proteins was detected in undifferentiated U‐937 cells. This constitutive level was increased time‐ and dose‐ dependently by incubation with phorbol myristate acetate (PMA). In U‐937 cells differentiated by 24 h incubation with 6 ng/ml PMA, dexamethasone (DEX) (1 μM for 16 h) caused an increased synthesis of the mRNA level of LCT‐1 and 2, but not of LCT‐5, over the level induced by PMA. DEX had no effect in undifferentiated cells, Moreover, DEX stimulated the extracellular release of LCT‐1 and 5, but not of LCT‐2, and inhibited the release of PGE2 and TXB2 only in the differentiated U‐937 cells. These results suggest that the responsiveness of these cells to glucocorticoids is dependent on the phase of cell differentiation. The selective release of lipocortins by differentiated U‐937 cells may explain, at least in part, the inhibition by DEX of the prostanoid release.
Purification, inhibitory properties and amino acid sequence of a new serine proteinase inhibitor from white mustard (Sinapis alba L.) seed
Authors: Menegatti Enea , Tedeschi Gabriella , Ronchi Severino , Bortolotti Fabrizio , Ascenzi Paolo , Thomas Richard M. , Bolognesi Martino , Palmieri Sandro
Published: July 2002

A new serine proteinase inhibitor, mustard trypsin inhibitor 2 (MTI‐2), has been isolated from white mustard (Sinapis alba L.) seed by affinity chromatography and reverse phase HPLC. The protein inhibits the catalytic activity of bovine β‐trypsin and bovine α‐chymotrypsin, with dissociation constants (K
d) of 1.6 × 10−10 M and 5.0 × 10−7 M, respectively, at pH 8.0 and 21°C, the stiochiometry of both proteinase‐inhibitor complexes being 1:1. The amino acid sequence of MTI‐2, which was determined following S‐pyridylethylation, is comprised of 63 residues, corresponding to a molecular weight of about 7 kDa, and shows only extremely limited homology to other serine proteinase inhibitors.
Ordered phosphorylation of p42mapk by MAP kinase kinase
Authors: Haystead Timothy A.J. , Dent Paul , Wu Jie , Haystead Clare M.M. , Sturgill Thomas W.
Published: July 2002

Preparation of milligram amounts of [32P]p42mapk, phosphorylaled at Tyr185 or diphosphorylated at Tyr185/Thr183, for use as specific protein phosphatase substrates is described. Tyr‐ but not Thr‐phosphorylated p42mapk, accumulates when ATP is limiting. Furthermore, Tyr185‐phosphorylated p42mapk exhibits an apparent 10‐fold decrease in apparent K
m (46.6 ± 6.6 nM) for MAP kinase kinase compared to that for the dephospho form (∼476 nM). We conclude that Tyr185 precedes Tyr185 phosphorylation, and that this is prerequisite, dramatically increasing the affinity of p42mapk for MAP kinase kinase.
SH3 — an abundant protein domain in search of a function
Authors: Musacchio Andrea , Gibson Toby , Lehto Veli-Pekka , Saraste Matti
Published: July 2002

Src‐homology 3 is a small protein domain of about 60 amino acid residues. It is probably made of β‐sheets. SH3 is present in a large number of eukaryotic proteins which are involved in signal transduction, cell polarization and membrane—cytoskeleton interactions. Here we review its occurrence and discuss possible functions of this domain.
Crystal structure of Pseudomonas aeruginosa apo‐azurin at 1.85 Å resolution
Authors: Nar Herbert , Messerschmidt Albrecht , Huber Robert , van de Kamp Mart , Canters Gerard W.
Published: July 2002

The 3D structure of apo‐azurin from Pseudomonas aeruginosa has been determined at 1.85 Å resolution. The crystal structure is composed of two different molecular forms of apo‐azurin arranged as hetero‐dimers in the tetramer of the asymmetric unit. Form 1 closely resembles the holo‐protein lacking copper. Form 2 shows differences in the metal binding site region induced by the incorporation of a solvent molecule into this site. The positions of the copper ligands His46 and His117 are shifted by 0.6 Å and 1.6 Å. The His117 side chain adopts a position at the surface of the protein, thereby facilitating access to the copper site. The presence of two different molecular forms of apo‐azurin in the crystal lattice may reflect an equilibrium between the two forms in solution. 1H‐NMR spectra or apo‐azurin recorded as a function or pH show that at high pH the line broadening of His35, His46 and His117 resonances is consistent with an interconversion between forms 1 and 2. At low pH, no broadening is observed. This may indicate that here the interconversion is fast on the NMR timescale.
Cooperative proton and calcium binding by sarcoplasmic reticulum ATPase
Authors: Martin R.Bruce
Published: July 2002

The classic work on binding of calcium to CaATPase is analyzed by an objective non‐linear least squares procedure of 74 data points over six pH values. Binding of two calciums to the basic form of the sites occurs with an equilibrium stability constant product of log K
1
K
2 = 13.2. Owing to competition from protons, this value drops in acidic and neutral solutions, becoming, for example, 11.9 at pH 6.8. Binding of the two calciums is so strongly cooperative that its extent is difficult to estimate reliably; there is very little of the one calcium species. Two protons are also bound cooperatively to the calcium sites. In solutions of calcium free protein, at pH < 7.6 the predominant species holds two protons at the calcium sites, while at greater pH the dominant species bears no protons; there is very little of the intermediate one proton species. The analysis also reveals the likely presence of small, less than statistical, amount of a ternary complex bearing one calcium and one proton.
Activity, disulphate mapping and structural modelling of the fifth domain of human β2‐glycoprotein I
Authors: Steinkasser Alexander , Barlow Paul N. , Willis Anthony C. , Kertesz Zsuzsa , Campbell Iain D. , Sim Robert B. , Norman David G.
Published: July 2002

Complexes formed by the interaction of negatively charged phospholipids and β2‐glycoprotein I(β2‐I) are the target of autoantibodies in systemic lupus erythematosus. The highly positively charged fifth (C‐terminal) domain of human β2‐I was produced as a fusion protein in an Escherichia coli expression system and was shown to bind to the negatively charged phospholipid, cardiolipin, almost as well as the intact protein. In an attempt to define the 3D structure of this domain, the disulphate linkage pattern was determined and shown to be Cys 1–4, Cys 2–5 and Cys 3–6 in contradiction to an earlier report. In the light of this information, the sequence of the fifth domain of β2I(β2‐I‐5) is readily aligned with that of the 16th repeat of factor H, of which the 3D structure is known, and a model of β2I‐5 has been built by homology. On the basis of the model we suggest residues that might be the target of profitable site‐directed mutagenesis in structure—function studies.
Glycogen synthase kinase‐3 and the Alzheimer‐like state of microtubule‐associated protein tau
Authors: Mandelkow E.-M. , Drewes G. , Biernat J. , Gustke N. , Van Lint J. , Vandenheede J.R. , Mandelkow E.
Published: July 2002

The Alzheimer‐like state of tau protein includes phosphorylation by a proline‐directed Ser/Thr kinase present in normal or pathological human brain. Extending earlier results on MAP kinase, we show here that the proline‐directed kinase, GSK3, can induce an Alzheimer‐like immune response involving several distinct and phoshorylatable epitopes at Ser—Pro motifs, as well as gel mobility shift, similar to MAP kinase. Both kinases behave like microtubule‐associated proteins in that they co‐purify through cycles of assembly and disassembly, and both kinases are directly associated with paired helical filaments.
Regulation by dexamethasone of P‐glycoprotein expression in cultured rat hepatocytes
Authors: Fardel Olivier , Lecureur Valérie , Guillouzo André
Published: July 2002

We have examined P‐glycoprotein (P‐gp) expression and function in cultured rat hepatocytes in response to dexamethasone (DEX), which is known to modulate various liver functions. Northern blot analyses revealed high levels of P‐gp mRNAs in cultured untreated liver cells in comparison to those found in freshly isolated hepatocytes, while DEX‐treated hepatoeytes also displayed elevated, although weaker, P‐gp levels. Similarly, Western blotting analysis indicated high levels of P‐gp in liver cells maintained in the absence of DEX. The use of mdr gene‐specific probes allowed us to show that DEX‐modulated P‐gp induction in cultured hepatocytes involved mostly, if not specifically, mdrl gene regulation. Doxorubicin P‐gp‐mediated efflux analyses revealed lower intracellular doxorubicin accumulation in DEX‐untreated liver cells than in DEX‐treated cells, thus indicating that over‐expressed P‐gp was functional. These data clearly show that DEX treatment strongly modulates P‐gp expression in primary rat hepatocyte cultures through a specific effect on the mdrl gene.
A rapid and efficient purification method for recombinant annexin V for biophysical studies
Authors: Burger A. , Berendes R. , Voges D. , Huber R. , Demange P.
Published: July 2002

Annexin V binds in a calcium‐dependent manner to acidic phospholipids and exhibits ion channel activity in vitro. We are investigating mutants of annexin V by single channel measurements, X‐ray crystallography and electron microscopy in order to understand the structure‐function relationships of the ion channel activity. We describe here a method to obtain very pure reeombinant annexin V required for such studies. The initial step is the mild opening of the bacterial cells by an osmotic shock. In the purification procedure, use is made of the reversible calcium‐mediated binding of annexin V to liposomes. In the last purification step the protein is subjected to ion‐exchange chromatography and elutes as a single peak free of any detectable contaminants.
Calcium‐mobilizing effectors inhibit P‐enolpyruvate carboxykinase gene expression in cultured rat hepatocytes
Authors: Valera Alfons , Solanes Gemma , Bosch Fatima
Published: July 2002

Incubation of primary cultures of hepatocytes from fed and fasted rats with calcium ionophore strongly decreased glucose production from pyruvate. Like insulin, calcium ionophore A23187, phenylephrine, vasopressin, and prostaglandins E2 and F2α caused a significant reduction (50–60%) in basal concentrations of mRNA for P‐enolpyruvate carboxykinase (PEPCK), the main regulatory enzyme of gluconeogenesis. Phenylephrine, prostaglandin E2 and calcium ionophore A23187 were also able to counteract the induction of PEPCK gene expression by Bt2cAMP. These effects were similar to those exerted by both vanadate and phorbol ester TPA. The decrease in extracellular calcium by the addition of the calcium‐chelating agent EGTA to the incubation medium caused an increase in PEPCK mRNA levels. This effect was additive to that of Bt2cAMP and was counteracted by vanadate.
Cyclosporine A protects mitochondria in an in vitro model of hypoxia/reperfusion injury
Authors: Gogvadze Vladimir , Richter Christoph
Published: July 2002

Hypoxia/reperfusion injury is a major clinical problem. One of its hallmarks is an increased cytosolic Ca2+ content and an increased generation of reactive oxygen species in the cytosol and in mitochondria. In the present study of an in vitro model of hypoxia/reperfusion injury, mitochondria are exposed to Ca2+ in combination with extra‐ and intramitochondrially acting prooxidants. In this model mitochondria are damaged in a Ca2+‐dependent manner. The extent and the site(s) of damage depend on both the kind of respiratory substrate and prooxidant used. The major damage occurs specifically at site I of the respiratory chain, and is due to hydrolysis of oxidized pyridine nucleotides and Ca2+ release followed by Ca2+ re‐uptake (Ca2+ ‘cycling’). Cyclosporine A completely protects against this damage. The protection is due to inhibition of pyridine nucleotide hydrolysis, an obligatory step in the sequence of events that links prooxidants to Ca2+ release from intact mitochondria.
One‐step purification and characterization of human pancreatic lipase expressed in insect cells
Authors: Thirstrup Kenneth , Carrière Frédéric , Hjorth Siv , Rasmussen Poul B. , Wöldike Helle , Nielsen Per F. , Thim Lars
Published: July 2002

A cDNA clone encoding the sequence of human pancreatic lipase (HPL) was subcloned into the baculovirus transfer vector pVL1392 and used in co‐transfection of Spodoptera frugiperda (Sf9) insect cells with wild‐type Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. A single recombinant protein (50 kDa) secreted by Sf9 cells was detectable in the culture medium 24 h post‐infection using both anti‐HPL polyclonal antibodies and potentiometric measurements of lipolytic activity. The expression level reached 40 mg/l of enzyme at 6 days. A single cation‐exchange chromatography was sufficient to obtain a highly pure recombinant HPL as demonstrated by N‐terminal sequencing, amino acid composition and carbohydrate analysis, as well as by mass spectrometry. These analyses revealed the production of mature protein with the correct processing of signal peptide and an homogenous glycosylation pattern. The kinetic properties of recombinant and native HPL were compared. Both enzymes showed similar profiles of interfacial activation, inhibition by bile salts and re‐activation by colipase.
Lysolipids reversibly inhibit Ca2+‐, GTP‐ and pH‐dependent fusion of biological membranes
Authors: Chernomordik Leonid V. , Vogel Steven S. , Sokoloff Aleksander , Onaran H.Ongun , Leikina Evgenia A. , Zimmerberg Joshua
Published: July 2002

Membrane fusion in exocytosis, intracellular trafficking, and enveloped viral infection is thought to be mediated by specialized proteins acting to merge membrane lipid bilayers. We now show that one class of naturally‐occurring phospholipids, lysolipids, inhibits fusion between cell membranes, organelles, and between organelles and plasma membrane. Inhibition was reversible, did not correlate with lysis, and could be attributed to the molecular shape of lysolipids rather than to any specific chemical moiety. Fusion was arrested at a stage preceding fusion pore formation. Our results are consistent with the hypothesis that biological fusion, irrespective of trigger, involves the formation of a highly bent intermediate between membranes, the fusion stalk.
Electron transfer in pheophytin a‐modified reaction centers from Rhodobacter sphaeroides (R‐26)
Authors: Shkuropatov A.Ya. , Shuvalov V.A.
Published: July 2002

The major part (> 90%) of bacteriopheophytin a in reaction centers (RCs) of Rhodobacter sphaeroides was substituted by plant pheophytin a. In modified RCs the photochemical formation of P+Qa
− occurs with with a quantum efficiency of 79%. The intermediary state P+I− displayed a recombination time constant of 1.5 ns, and the electron transfer from I− to Qa was characterized by a time constant of 540 ps. On the basis of spectral properties of P+I− for native and modified RCs, it was suggested that bacteriopheophytin, as well as bacteriochlorophyll monomers located in L protein branch, have a transition at 545 nm with approx. equal extinction coefficients. Accordingly, the state P+I− in modified RCs is proposed to consist of a thermodynamic mixture of P+BL− (∼ 80%) and P+Phe− (∼ 20%).
How does the switch II region of G‐domains work?
Authors: Stouten Pieter F.W. , Sander Chris , Wittinghofer Alfred , Valencia Alfonso
Published: July 2002

The transition of guanine nucleotide binding proteins between the ‘on’ (GTP‐bound) and ‘off’ (GDP‐bound) states has become a paradigm of molecular switching after a chemical reaction. The mechanism by which the switch signal is transmitted to the downstream recipients in the intracellular signal pathway has been extensively studied by biochemical, biophysical and genetic methods, but a clear picture of this process has yet to emerge. Based on the similarities of ras‐p21 and elongation factor Tu we propose here a model of the GDP state of ras‐p21 that is in agreement with all relevant experimental evidence. The model provides important clues about: (1) a possible molecular mechanism for signal transmission from the site of GTP hydrolysis to downstream effectors; (2) a major conformational change during signal generation and a key residue involved in this process (Tyr‐64); and (3) regions in ras‐p21 that can be differentially recognized by binding to external partners in a GTP/GDP state dependent fashion, most notably residues D69, Q70, R73, T74, R102, K104, D105 at the end of the α‐helices 2 and 3.
The protein import receptor MOM19 of yeast mitochondria
Authors: Moczko Martin , Gärtner Frank , Pfanner Nikolaus
Published: July 2002

We have identified the protein import receptor MOM19 of Saccharomyces cerevisiae mitochondria. MOM19 is exposed on the outer membrane surface and present in the mitochondrial receptor complex. Antibodies raised against MOM19 strongly inhibited the import of preproteins into isolated yeast mitochondria. Fab fragments prepared from the antibodies showed the same inhibitory effect. By using mutant mitochondria, which lacked the second import receptor MOM72, we found that the import of preproteins via MOM19 did not require the presence of MOM72. We conclude that MOM19 is required for preprotein translocation across the yeast mitochondrial outer membrane and is able to function independently of the receptor MOM72.
Maltose transport system of Escherichia coli: An ABC‐type transporter
Authors: Nikaido Hiroshi
Published: July 2002

The maltose transport system of E. coli is composed of a periplasmic maltose‐binding protein (MBP), the presumed transmembrane channel made up of MalF and MalG proteins, and two copies of the ATPase subunit, MalK. The membrane‐associated transporter complex was purified in a functional form both from the wild‐type strain and from mutants that do not require MBP for transport, and was reconstituted into proteoliposomes. A major function of MBP is to send a transmembrane signal, in the presence of ligands, to the ATPase subunits on the inner side of the membrane. In addition, MBP performs a special function in the translocation of the larger ligands, maltodextrins, perhaps by aligning them for entry into the channel.