bluuebunny/crossref_metadata_2025 huggingface dataset:
Conformational disorder of the distal leucine in monomeric Glycera hemoglobins and implications for oxygen binding
Authors: Cooke Robert M. , Wright Peter E.
Published: July 2002

1H NMR studies of the carbon monoxide complexes of the major monomeric hemoglobins from Glycera dibranchiata show that distal leucine is conserved at position E7. The observed ring current shifts and nuclear Overhauser enhancements indicate conformational disorder of the leucine E7 side chain. The conformational substates interconvert rapidly on the NMR time scale. The rapid conformational fluctuations of leucine E7 may play a fundamental role in governing diffusion of ligands to the heme.
Protein fatty acyltransferase is located in the rough endoplasmic reticulum
Authors: Berger Michael , Schmidt Michael F.G.
Published: July 2002

The fatty acid acylation of polypeptides was studied in vivo and in vitro by incorporation of radiolabeled palmitic acid into Semliki Forest viral polypeptides. Utilizing a cell‐free system for acylation protein fatty acyltransferase was characterized as an integral membrane protein. No acylation activity was detected in the cytosol. During subcellular fractionation of a variety of mammalian or avian cells the enzyme was localized to the rough endoplasmic reticulum. Therefore this posttranslational hydrophobic modification starts earlier in the biosynthesis of acylated polypeptides than previously believed.
Evidence for a 5(6)‐epoxytetraene intermediate in the biosynthesis of lipoxins in human leukocytes
Authors: Puustinen Tapio , Webber Stephen E. , Nicolaou K.C. , Haeggström Jesper , Serhan Charles N. , Samuelsson Bengt
Published: July 2002

The existence of a 15(S)‐hydroxy‐5,6‐oxido‐7,9,13‐trans 11‐cis‐eicosatetraenoic acid intermediate in the biosyntheses of lipoxins A and B has recently been proposed. In the present study, human leukocytes were exposed to 15‐HETE and the divalent cation ionophore A23187 and alcohol trapping studies were performed. The products containing alkyltetraenes were isolated and characterized. HPLC analysis, UV spectroscopy and GC/MS of the products showed that 5,15‐dihydroxy‐14‐O‐alkyleicosatetraenoic acids were formed, indicating that 5(6)‐epoxytetraenes (precursor of the trapping product) were formed in human leukocytes. To gain further evidence for the role of 5(6)‐epoxytetraene intermediate in the biosynthesis of lipoxins, (15)‐hydroxy‐5,6‐oxido‐ 7,9,13‐trans‐11‐cis‐eicosatetraenoic acid was prepared by total chemical synthesis. When added to purified human liver cytosolic epoxide hydrolase, the epoxide was rapidly and quantitatively converted into LXA. The results provide further evidence for the role of a 5(6)epoxytetraene intermediate in the biosynthesis of lipoxins.
Bacillus thuringiensis toxin inhibits K+‐gradient‐dependent amino acid transport across the brush border membrane of Pieris brassicae midgut cells
Authors: Sacchi V.Franca , Parenti Paolo , Hanozet Giorgio M. , Giordana Barbara , Lüthy Peter , Wolfersberger Michael G.
Published: July 2002

The luminal membrane of larval midgut cells is the site of action of insecticidal delta‐endotoxin from Bacillus thuringiensis. At concentrations that correspond to normal effective doses in vivo, the toxin inhibits the uptake of amino acids by brush border membrane vesicles prepared from midguts of Pieris brassicae larvae. The toxin does not interact with the K+‐amino acid symport but rather increases the K+ permeability of the membrane. The toxin does not increase the permeability of lepidopteran midgut brush border membrane to either Na+ or H+ nor does it increase the K+ permeability of brush border membrane vesicles prepared from mammalian small intestine,
Senescence‐associated decrease of NADPH‐induced lipid peroxidation in rat liver microsomes
Authors: Devasagayam Thomas P.A.
Published: July 2002

Senescence is associated with decrease in the NADPH‐induced lipid peroxidation in liver homogenate as well as rough and smooth microsomes of female rats. In the microsomal fractions, sensitivity to NADPH‐induced lipid peroxidation is high in young adults (3‐month‐old), decreases in middle aged (12‐month‐old) and reaches lowest levels in senescent (30‐month‐old) rats. Increasing the concentration of co‐factors or time of incubation does not alter this resistance observed in the senescent rats. Major factors responsible for this resistance in senescent rats seem to be low levels of substrate in the form of phospholipids, NADPH‐cytochrome c reductase, cytochrome P‐450 and high cholesterol:phospholipid ratios besides enhanced levels of Superoxide dismutase, α‐tocopherol and reduced glutathione.
β‐oxidation of polyunsaturated fatty acids in peroxisomes Subcellular distribution of Δ3,Δ2‐enoyl‐CoA isomerase activity in rat liver
Authors: Kärki T. , Hakkola E. , Hassinen I.E. , Hiltunen J.K.
Published: July 2002

The metabolism of the double bonds at the Δ3 position in fatty acids was studied in rat liver. Infusion of δ3‐trans‐dodecenoic acid into isolated perfused liver and subecllular fractionation studies showed the presence of both peroxisomal and mitochondrial Δ3,Δ2‐enoyl‐CoA isomerase activity (EC 5.3.3.8). These findings together with the previous demonstration of peroxisomal 2,4‐dienoyl‐CoA reductase (EC 1.3.1.34) [(1981) J. Biol. Chem. 256, 8259–8262] and D‐3‐OH‐acyl‐CoA epimerase (EC 5.1.2.3) [(1985) FEBS Lett. 185, 129–134] activities show that peroxisomes possess all the auxiliary enzymes required for the β‐oxidation of unsaturated fatty acids.
Differential effects of DNA tumor virus nuclear oncogene products on adipocyte differentiation
Authors: Tobe Kazuyuki , Kasuga Masato , Kitasato Hidero , Takaku Fumimaro , Takano Toshiya , Segawa Kaoru
Published: July 2002

We have introduced SV40 and polyoma large T antigen‐ and adenovirus‐type 12 E1A genes into mouse 3T3‐L1 preadipocyte cells to study the ability of various nuclear oncogene products to modulate cell differentiation. Clones expressing E1A products could differentiate into adipocytes faster than the control in spite of the absence of adipogenic inducers, as measured by the appearance of lipid droplets microscopically and by staining accumulated triglycerides with oil red O. However, clones expressing SV40 and polyoma large T antigens could not differentiate even if they were exposed to the inducers.
Oxidation‐reduction midpoint potentials of the molybdenum center in spinach NADH:nitrate reductase
Authors: Barber Michael J. , Notton Brian A. , Solomonson Larry P.
Published: July 2002

Oxidation‐reduction midpoint potentials for the molybdenum center in assimilatory NADH:nitrate reductase isolated from spinach (Spinacia oleracea) have been determined at pH 7.0 in the presence of dye mediators using EPR spectroscopy to monitor formation of Mo(V). Values for the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) couples were determined to be −8 and −42 mV, respectively.
Nucleotide and primary sequence of a major rice prolamine
Authors: Kim Woo Taek , Okita Thomas W.
Published: July 2002

A recombinant cDNA clone encoding a major rice seed storage prolamine was isolated by antibody screening of a cDNA λgt 11 library. This clone contained a single open reading frame encoding a putative rice prolamine precursor (M
r17 300). In contrast to other cereal prolamines, the primary sequence of the rice prolamine was devoid of any major tandem repetitive sequences, a feature prevalent in all cereal prolamines studied to date. No significant homology was detected between the rice prolamine and other cereal prolamines, indicating that the rice gene evolved from unique ancestral DNA segments.
Nerve growth factor increases the cyclic GMP level and activates the cyclic GMP phosphodiesterase in PC12 cells
Authors: Laasberg Tiit , Pihlak Arno , Neuman Toomas , Paves Heiti , Saarma Mart
Published: July 2002

Nerve growth factor (NGF) rapidly increases the cyclic GMP (cGMP) level about 2–3‐fold and enhances the cGMP phosphodiesterase (PDE) activity about 2‐fold in rat pheochromocytoma PC12 cells. No changes in the level of cyclic AMP (cAMP) and in the activity of cAMP PDE were found. GTP and a nonhydrolysable analog of GTP, GMP‐PCP, at 100 μM, were able to mimic the effect of NGF on the cGMP PDE activity. These results suggest that the cGMP system may be one of the second messengers of NGF action in PC12 cells.
Architectonics of a bacterial flagellin filament subunit
Authors: Fedorov O.V. , Kostyukova A.S. , Pyatibratov M.G.
Published: July 2002

Flagellins of two Escherichia coli strains and their tryptic fragments were studied by different methods. Probabilities of secondary structure formation were also calculated for all flagellins with a known primary structure. The obtained data permit one to suggest a model for the flagellin molecule consisting of a central part responsible for antigenic properties and terminals responsible for polymerization. The central part is variable in length from a few amino acid residues to three‐four hundred depending on the bacterial species. The terminal parts consist of about 160 amino acid residues from the N‐end and 100 from the C‐end.
Calcium mobilization by angiotensin II and neurotransmitter receptors expressed in Xenopus laevis oocytes
Authors: Sandberg Kathryn , Markwick Alison J. , Trinh Dorothy P. , Catt Kevin J.
Published: July 2002

Specific receptors for angiotensin II (AII) were expressed in albino Xenopus laevis oocytes co‐injected with poly(A)+ mRNA isolated from rat adrenal cortex and the calcium‐specific photoprotein, aequorin. In such oocytes, AII elicited rapid, dose‐dependent rises in cytosolic free calcium with light emission responses up to 100‐fold above basal levels. Ligand‐induced light emission was also observed in oocytes injected with rat brain mRNA and stimulated with acetylcholine and glutamate. These findings demonstrate that mammalian AII receptors expressed in Xenopus oocytes are functionally linked to intracellular Ca2+ mobilization, and indicate the potential value of aequorin‐injected oocytes for rapid and specific screening of mRNAs transcribed from expression libraries containing cloned receptor cDNAs.
Isolation and electrophoretic analysis of surface proteins of the zygomycete Absidia glauca
Authors: Teepe Hildegard , Böttge Jörg-Andreas , Wöstemeyer Johannes
Published: July 2002

Methods for isolation and electrophoretic analysis of surface proteins from the mucoraceous fungus Absidia glauca are described. By analysing the set of cell wall associated proteins which can be extracted with 1 M LiCl, we could show marked differences between submersed and aerial mycelium. Furthermore, during differentiation of the aerial mycelium protein patterns change remarkably. We were able to demonstrate the existence of mating type specific proteins, the most prominent of which is a 15 kDa polypeptide expressed in submersed as well as in aerial hyphae of the (+) mating type.
Further evidence for the presence of a thiazoline ring in the isoleucylcysteine dipeptide intermediate in bacitracin biosynthesis
Authors: Ishihara Hiroaki , Shimura Kensuke
Published: July 2002

Isoleucylcysteine dipeptide, a first intermediate peptide in bacitracin biosynthesis, was liberated from the enzyme protein and oxidized with manganese dioxide in dimethylsulfoxide. The resulting oxidation product was identified by thin‐layer chromatography as 2‐(2‐methyl‐l‐oxobutyl)‐thiazole‐4‐carboxylic acid which has been isolated from the hydrolysate of bacitracin F. This result shows that the intermediate dipeptide contains a thiazoline ring, and that the thiazoline ring is synthesized at the dipeptide stage in the process of peptide chain elongation in bacitracin biosynthesis. Improbability of non‐enzymatic dehydrative cyclization of the dipeptide is discussed.
Polypeptide constitution of receptors for apamin, a neurotoxin which blocks a class of Ca2+‐activated K+ channels
Authors: Auguste Patrick , Hugues Michel , Lazdunski Michel
Published: July 2002

Affinity labelling experiments with different azido‐125I‐apamin derivatives were carried out to identify polypeptide components of the apamin‐sensitive Ca2+‐activated K+ channel in brain and pheochromocytoma cell membranes. Different polypeptides were labelled with different apamin derivatives. The major component has a molecular mass of about 30 kDa but other components at 45, 58 and 86 kDa were also identified. Results obtained with brain membranes on one hand and pheochromocytoma cells on the other were not exactly identical and suggest that there are sub‐types of apamin receptors.
Light‐stimulated phosphorylation of proteins in cell‐free extracts from Trichoderma viride
Authors: Grešík Miroslav , Kolarova Nadežda , Farkaš Vladimír
Published: July 2002

When illuminated by visible light, cell‐free extracts from the fungus Trichoderma viride catalysed the phosphorylation of at least two proteins with molecular masses of 18 and 114 kDa which were practically absent when the phosphorylation was performed in the dark. The effect of light could be substituted by 3mM cyclic AMP, not only in the cell‐free extract, but also in the separated cytosol. It is concluded that the process of photoinduced conidiation in Trichoderma involves phosphorylation of conidiation‐specific proteins by (a) cyclic AMP‐dependent protein kinase(s) present in the cytosol.
Complete assignment of the 1H NMR spectrum of a synthetic zinc finger from Xfin Sequential resonance assignments and secondary structure
Authors: Lee Min S. , Cavanagh John , Wright Peter E.
Published: July 2002

A 25‐residue synthetic peptide corresponding to zinc finger 31 of the Xenopus protein Xfin adopts a compact, folded conformation in the presence of zinc. Complete 1H resonance assignments have been made. The peptide contains a helix, beginning as an α‐helix and ending as a 310‐helix, that extends from residue 12 to 23. Several positively charged and polar residues located on this helix are likely to be involved in interactions with DNA. Residues 1–10 appear to adopt a hairpin‐like structure.
Purification and partial characterization of rat liver soluble catechol‐ O‐methyltransferase
Authors: Tilgmann Carola , Kalkkinen Nisse
Published: July 2002

The rat liver soluble catechol‐O‐methyltransferase (EC 2.1.1.6.) has been purified utilizing a combination of conventional chromatography and HPLC. The purified enzyme has a molecular mass of 25 kDa, a pI of 5.1, and exists in two forms which differ in the nature of their intramolecular disulfide bonds. This difference causes these two protein forms to behave differently in reversed phase chromatography.
Signal transduction in human monocytes and granulocytes through the PI‐linked antigen CD14
Authors: Lund-Johansen Fridtjof , Olweus Johanna , Aarli Ånen , Bjerknes Robert
Published: July 2002

A possible role for the Pi‐linked CD14 molecule in human monocyte and granulocyte signal mediation was investigated. Using flow cytometry and the fluorescent indicators Fluo‐3 and dihydrorhodamine‐123 it was shown that crosslinking of the CD14 molecule induces an increase in monocyte and granulocyte cytoplasmic calcium concentration and monocyte H2O2 production. These responses were found to be independent of IgG Fc receptors and suggest an intrinsic signal mediating capacity of the CD 14 molecule.
A high throughput assay for inhibitors of HIV‐1 protease Screening of microbial metabolites
Authors: Sarubbi Edoardo , Nolli M.Luisa , Andronico Franca , Stella Sergio , Saddler Gerard , Selva Enrico , Siccardi Antonio , Denaro Maurizio
Published: July 2002

A novel method for discovery of HIV‐1 protease inhibitors in complex biological samples has been developed. The assay is based on two specific reagents: a recombinant protein constituted by a portion of the HIV‐1 Gag polyprotein comprising the p17–p24 cleavage site, fused to E. coli β‐galactoxidase, and a monoclonal antibody which binds the fusion protein in the Gag region. Binding occurs only if the fusion protein has not been cleaved by the HIV‐1 protease. The assay has been adapted for the screening of large numbers of samples in standard 96‐well microliter plates. Using this method about 12000 microbial fermentation broths have been tested and several HIV‐1 protease inhibitory activities have been detected. One of these has been studied in detail.
Zinc is required for folding and binding of a single zinc finger to DNA
Authors: Lee Min S. , Gottesfeld Joel M. , Wright Peter E.
Published: July 2002

A synthetic peptide corresponding to zinc finger 31 of the Xenopus protein adopts a folded conformation in the presence of zinc. The same peptide in the absence of zinc is not folded in a stable tertiary conformation, as determined by NMR. Binding experiments have shown that the peptide binds non‐specifically to DNA only in the presence of zinc. Moreover, competive DNA binding experiments indicate interaction with 3.9 ± base pairs.
Fourier transform infrared spectroscopic study on retinochrome and its primary photoproduct, lumiretinochrome
Authors: Sekiya Noriko , Kishigami Akio , Naoki Hideo , Chang Chia-Wung , Yoshihara Kazuo , Hara Reiko , Hara Tomiyuki , Tokunaga Fumio
Published: July 2002

Structural studies of retinochrome, and its photoproduct, lumiretinochrome, were done by Fourier transform infrared difference spectroscopy. The absorption bands in the carbonyl stretching region which shift in D2O show the changes in the protein part during the photoreaction. Strong absorption bands in the finger‐print region show that the all‐trans‐retinal chromophore in retinochrome isomerizes to the 11‐cis‐retinal chromophore in lumiretinochrome upon illumination with yellow‐green light at 83K.
Primary structure of the virus activating protease: from chick embryo Its identity with the blood clotting factor Xa
Authors: Suzuki Harukazu , Harada Atzuko , Hayashi Yumiko , Wada Kohji , Asaka Jun-ichiro , Gotoh Bin , Ogasawara Tomohiko , Nagai Yoshiyuki
Published: July 2002

Host cell proteases activating, para‐ and orthomyxovirus fusion glycoprotein precursors play a crucial role in determining the viral tropism in infected organisms, We previously isolated such an endoprotease from the allantoic fluid of chick embryo and showed its close similarity to the activated form of blood clotting factor X (FXa) by partial amino acid sequencing. In this report, we have cloned and sequenced a cDNA of the protease and show that it is encoded in a single gene as a preproform with all the functional and structural domains known to be characteristic of bovine or human FX, establishing the identity between the protease and FXa.
Protein secondary structure determination by NMR Application with recombinant human cyclophilin
Authors: Wüthrich Kurt , Spitzfaden Claus , Memmert Klaus , Widmer Hans , Wider Gerhard
Published: July 2002

It is a unique trait of the NMR method for protein structure determination that a description of the polypeptide secondary structure can be obtained at an early stage and quite independently of the complete structure calculation. In this paper the procedures used for secondary structure determination are reviewed and placed in perspective relative to the other steps in a complete three‐dimensional structure determination. As an illustration the identification of the regular secondary structure elements in human cyclophilin is described.
Transcriptional control of yeast phosphofructokinase gene expression
Authors: Heinisch J. , Vogelsang K. , Hollenberg C.P.
Published: July 2002

We here provide the complete nucleotide sequences of the 5′‐non‐coding regions of the yeast phosphofructokinase genes, PFK1 and PFK2. lacZ fusions of the PFK1 and PFK2 promoters were constructed and a deletion analysis was performed. In contrast to other glycolytic gene promoters, no strong regulatory elements could be found. However, we detected moderate UAS and URS functions. In general, the effects on expression upon deletion of these regions were more pronounced on media containing ethanol than on those containing glucose as carbon sources. Overexpression of either one of the PFK genes led to a decreased enzymatic activity in a wild‐type background but did not affect transcription from the promoters.
Reconstitution of active sucrose transport in plant proteoliposomes
Authors: Li Ze-Sheng , Gallet Olivier , Gaillard Cécile , Lemoine Rémi , Delrot Serge
Published: July 2002

The proteins of purified plasma membranes from sugar beet (Beta vulgaris L.) leaf were solubilized and separated on a size exclusion column. The fractions eluted from the column were monitored by ELISA with antibodies directed to a putative sucrose carrier protein. The peak most reactive in ELISA was approximately 120 kDa, and yielded a 40 kDa peak after denaturation by SDS. The 120‐kDa peak was recovered and used for reconstitution experiments with asolectin. Upon imposition of an artificial pH gradient and electrical gradient, the obtained proteoliposomes exhibited active transport of sucrose, but not of valine. The active transport of sucrose was inhibited by N‐ethylmaleimide and HgCl2.
Acetylcholine interactions with tryptophan‐184 of the α‐subunit of the nicotinic acetylcholine receptor revealed by transferred nuclear Overhauser effect
Authors: Fraenkel Yigal , Gershoni Jonathan M. , Navon Gil
Published: July 2002

Acetylcholine interactions with three genetically engineered fusion proteins containing peptides from the nicotinic acetylcholine receptor were studied by ID and 2D nuclear magnetic resonance methods. The three proteins were Torpedo α186–200, Torpedo α186–198, and human α183–204 of the acetylcholine receptor fused to the first 323 residues of the E. coli protein trpE. Nuclear Overhauser effect studies revealed interactions of bound acetylcholine with tryptophan‐184 present in the Torpedo α184–200, and the human α183–204 sequences. These interactions are between the N(CH3)3
+ and CH3 groups of acetylcholine with the aromatic protons of tryptophan. The appearance of these cross‐peaks indicates a distance of less than 5 A between tryptophan and the bound ligand; however, direct contact has yet to be proven.
Kinetic mechanism of pyruvate decarboxylase Evidence for a specific protonation of the enzymic intermediate
Authors: Ermer Joachim , Schellenberger Alfred , Hübner Gerhard
Published: July 2002

Decarboxylation of pyruvate by pyruvate decarboxylase (EC 4.1.1.1) was performed in a reaction mixture containing 50% deuterium. The isolated product, acetaldehyde, was investigated directly by 1H NMR and by mass spectrometry after conversion to the 2,4‐dinitrophenyl hydrazone. The protium content of 56% at acetaldehyde Cl demonstrate a specific protonation of the corresponding intermediate by the enzyme. Proton inventory studies and enzyme modification indicate the 4′ amino group of the coenzyme, thiamine pyrophosphate, in an immonium structure being a possible proton donor. A ‘partially concerted’ mechanism is suggested for the reaction steps following the decarboxylation.
Puromycin reaction for the A site‐bound peptidyl‐tRNA
Authors: Semenkov Yu. , Shapkina T. , Makhno V. , Kirillov S.
Published: July 2002

AcPhe2‐tRNAPhe synthesized in 70S ribosomes after consecutive binding of AcPhe‐tRNAPhe at the P sites and EF‐Tu‐directed binding of Phe‐tRNAPhe at the A sites is able to react quantitatively with puromycin in the absence of EF‐G. A detailed study of the kinetics of the puromycin reaction, its comparison with that of spontaneous translocation, the use of antibiotic viomycin as an effective inhibitor of spontaneous translocation revealed that, besides spontaneous translocation, this peptidyl‐tRNA could react with puromycin being located at the A site. This leads to the conclusion that the transpeptidation reaction per se triggers conformational changes in the ribosomal complex bringing the 3′‐end of a newly synthesized peptidyl‐tRNA nearer to the peptidyl‐site of the peptidyltransferase center. This is detected functionally as the ability of such an A site bound peptidyl‐tRNA to react with puromycin. This reaction is highly pronounced at elevated (25°C) temperature but can be hardly detected at 0°C.
Lethal oxidative damage to human immunodeficiency virus by human recombinant myeloperoxidase
Authors: Moguilevsky Nicole , Steens Martine , Thiriart Clotilde , Prieels Jean-Paul , Thiry Lise , Bollen Alex
Published: July 2002

Human recombinant myeloperoxidase was evaluated in a cell‐free system for its inactivation properties on the replication of human immunodeficiency virus, HTLV‐IIIB. In the presence of a hydrogen peroxide generating system (glucose and glucose oxidase) and sodium thiocyanate, the recombinant enzyme inhibited virus‐induced syncytium formation and viral replication without causing any cytopathic effects on SupT1 reporter cells. In addition, U937 monocytoid cells, chronically infected with HIV1, were exposed to recombinant myeloperoxidase (10 U/ml) and monitored during 48 h for the accumulation of intracellular p24 viral antigen. Under these conditions, the recombinant enzyme significantly reduced intracellular viral replication without affecting cell viability.
Elucidation of the gene defect in Marfan syndrome Success by two complementary research strategies
Authors: Peltonen Leena , Kainulainen Katariina
Published: July 2002

Marfan syndrome, which is characterized by manifestations in the skeletal, ocular and cardiovascular systems, is one of the most common inherited connective‐tissue disorders. The independently performed genetic assignment of the Marfan locus and classical biochemical and immunohistochemical analyses complemented each other in the search for the Marfan gene defect and in 1991 the fibrillin gene in chromosome 15 was identified as the Marfan gene. So far, three mutations leading to the Marfan phenotype have been reported in this gene coding for a microfibrillar protein. The available data suggests a wide spectrum of different mutations of fibrillin and although mutations of the fibrillin gene account for the majority of Marfan cases, evidence also exists for locus heterogeneity in a minority of Marfan cases.
Human Interleukin‐1 receptor antagonist High yield expression in E. coli and examination of cysteine residues
Authors: Steinkasserer Alexander , Solari Roberto , Mott Helen R. , Aplin Robin T. , Robinson Carol C. , Willis Anthony C. , Sim Robert B.
Published: July 2002

The human IL‐1 receptor antagonist (IL‐1ra) was produced in a high yield E. coli expression system, and was purified in a rapid two‐step purification, This recombinant IL‐1ra molecule possessed full binding activity to the IL‐1 receptor (type I) and totally inhibited IL‐1‐induced PGE2 production by human dermal fibroblasts. Radioalkylation and analysis of V8‐derived IL‐1ra peptides indicate that the four cysteines present in the IL‐1ra are not disulphide‐linked.
Participation of the overproduced elongation factor Tu fromThermus thermophilusin protein biosynthesis ofEscherichia coli
Authors: Zeidler Waltraud , Kreutzer Roland , Sprinzl Mathias
Published: July 2002

The influence of the overproduced elongation factor Tu (EF‐Tu) fromThermus thermophiluson the protein biosynthesis inEscherichia coliwas investigated both in vivo and in vitro. A kirromycin‐resistantE. colistrain became sensitive to this antibiotic upon the expression of thetufA‐gene ofT. thermophiluspresent on a plasmid. In in vitro translation with components of the kirromycin‐resistantE. colistrain the poly(Phe) synthesis stopped when minute amounts of the EF‐Tu fromT. thermophiluswere added. Both results indicate the sensitivity of theT. thermophilusEF‐Tu to kirromycin and its participation in the polypeptide synthesis ofE. coli.
Expression of glutathione S‐transferase P‐form in primary cultured rat liver parenchymal cells by coplanar polychlorinated biphenyl congeners is suppressed by protein kinase inhibitors and dexamethasone
Authors: Aoki Yasunobu , Matsumoto Michi , Suzuki Kazuo T.
Published: July 2002

Glutathione S‐transferase P‐form (GST‐P, EC 2.5.1.18) mRNA was expressed by epidermal growth factor as well as by 3,4,5,3',4'‐penta‐chlorinated biphenyl (PenCB) in primary cultured rat liver parenchymal cells. The expression of GST‐P was suppressed by inhibitors of protein kinase C and dexamethasone, an antagonist of AP‐1 transcription factor activity, whereas expression of cytochrome P4501A2 by PenCB was not affected by these reagents. The AP‐1 related transcription factor may be essential for the expression of GST‐P by PenCB as also may be a protein kinase C type enzyme.
Activation of muscarinic receptors increases the concentration of free Na+ in mouse pancreatic B‐cells
Authors: Gilon P. , Henquin J.C.
Published: July 2002

The fluorescent probe SBFI was used to monitor the influence of acetylcholine (ACh) on the cytosolic concentration of free Na+ (Na+
i) in single mouse pancreatic B‐cells. In the presence of 3 mM glucose and 135 mM extracellular Na+, Na+
i, averaged 16.6 mM. ACh (100μM) increased Na+
i by ~ 80%. This rise was prevented by atropine, a blocker of muscarinic receptors, and by omission of extracellular Na+, but still occurred if the sodium pump was blocked by ouabain. It was unaffected by tetrodotoxin, a blocker of voltage‐sensitive Na+ channels, and was not mimicked by depolarization of the cells with high K+ It is concluded that activation of muscarinic receptors increases the membrane permeability to Na+ in pancreatic B‐cells.
Presence of the gypsy (MDG4) retrotransposon in extracellular virus‐like particles
Authors: Syomin Boris V. , Kandror Konstantin V. , Semakin Andrej B. , Tsuprun Vladimir L. , Stepanov Alexander S.
Published: July 2002

As a first step to investigate the functional activity of gypsy virus‐like particles (VLPs) we explored the possibility of preservation of its VLP in extracellular form. The preparations containing extracellular gypsy VLP from Drosophila melanogaster and D. virilis were obtained. Full‐length polyA+ RNA and polyA+ RNA‐DNA complexes of gypsy were observed in both preparations. The polypeptides with some specificity to gypsy nucleic acids were identified in the obtained VLP preparations. These data accompanied by morphological characteristics of samples testify the presence of intact gypsy VLP in cultured media both from D. melanogaster and D. virilis cultivated cells.
Immunocytochemical analyses of annexin V (CaBP33) in a human‐derived glioma cell line
Authors: Giambanco Ileana , Sorci Guglielmo , Antonioli Sabrina , Rambotti Maria Grazia , Spreca Antonio , Bocchini Virginia , Donato Rosario
Published: July 2002

The subcellular distribution of annexin V, a calcium‐dependent phospholipid‐ and membrane‐binding protein, in a human‐derived cell line, GL15, was investigated by immunocytochemistry at light and electron microscope levels. Annexin V was found diffusely in the cytoplasm and associated with plasma membranes, membranes delimiting cytoplasmic vacuoles, membranes of the endoplasmic reticulum, and filamentous structures the identity of which remains to be established. By immunocytochemistry at the light microscope level and immunochemistry, the expression of annexin V in these cells was found to depend on cellular growth state, being maximal soon after plating and progressively declining thereafter. However, re‐expression of annexin V was observed whenever cell proliferation slowed down or arrested. These findings suggest that annexin V in glioma cells is mostly expressed in connection with cell differentiation. Also, the present ultrastructural data suggest that plasma membranes, membranes of the endoplasmic reticulum and the cytoskeleton are prominent sites of action of annexin V in vivo, thus lending support to the possibility that this protein might have a role in the regulation of cytoskeleton elements and/or of the structural organization of membranes.
Three‐dimensional structure and antigenicity of transmembrane‐protein peptides of the human immunodeficiency virus type 1
Authors: Klasse Per Johan , Pipkorn Rüdiger , Blomberg Jonas , Han Kyou-Hoon , Hilton Bruce , Ferretti James A.
Published: July 2002

A point mutation (Ala‐589 to Thr) in the transmembrane protein of the human immunodeficiency virus type 1 (HIV‐1) has been shown to decrease the sensitivity of the virus to the neutralizing effect of human HIV‐1 specific antibodies [(1990) J. Virol. 64, 3240‐3248]. Here 17‐residue peptides with the parental and mutant sequences were compared: the parental peptide bound antibodies of sera from HIV‐1 infected persons more frequently and with higher affinity than the mutant peptide. However, according to circular dichroism (CD), NMR spectroscopy and molecular modelling the peptides have indistinguishable backbone conformations under a variety of experimental conditions. These techniques showed for both peptides that no ordered helix was present in water solution. However, for both peptides in alcohol‐water solutions approximately 60% α‐helix coula be induced. The three‐dimensional structures of these peptides provide a basis for understanding how this mutation in the transmembrane protein may affect the interaction with both the outer envelope glycoprotein and with antibodies.
Thiol dependent isomerization of all‐trans‐retinoic acid to 9‐cis‐retinoic acid
Authors: Urbach Jonathan , Rando Robert R.
Published: July 2002

The important biological effector 9‐cis‐retinoic acid can be generated by liver microsomes or by bovine serum albumin in detergent. The mechanism of this isomerization reaction is the subject of these studies. The protein mediated isomerization process is shown to be thiol‐ and pH‐dependent. Moreover, the retinoic acids are also isomerized by 1‐dodecanethiol in the presence of detergents. This isomerization process is pH‐dependent as well, with isomerization rates increasing with pH. The isomerization reactions are quenched with free radical traps, such as α‐tocopherol and ascorbic acid, suggesting that a thiol radical mechanism, rather than a thiolate anion‐dependent mechanism, is implicated here. The pH dependence can be understood in terms of a thiol radical mechanism, because thiol radicals are produced from thiolate anions in the presence of oxygen. The facile thiol‐mediated isomerization of the retinoic acids suggests that this could be a physiologically relevant mechanism for the formation of 9‐cis‐retinoic acid from all‐trans‐retinoic acid.
Substitution of conserved tyrosine residues in helix 4 (Y143) and 7 (Y293) affects the activity, but not IAPS‐forskolin binding, of the glucose transporter GLUT4
Authors: Wandel Sonja , Schürmann Annette , Becker Walter , Summers Scott A. , Shanahan Michael F. , Joost Hans G.
Published: July 2002

Six tyrosine residues (Y28, Y143, Y292, Y293, Y308, Y4321) which are conserved in all mammalian glucose transporters were substituted for phenylalanine by site‐directed mutagenesis, and mutant glucose transporters were transiently expressed in COS‐7 cells. Glucose transport activity as assessed by reconstitution of the solubilized transporters into lecithin liposomes was reduced by 70% in the mutant Y143F and appeared to be abolished in Y293F, but was not affected by substitution of Y28, Y292, Y308 and Y432. In contrast, covalent binding of the photolabel 125IAPS‐forskolin was normal in all mutants. Stable expression of the mutants Y143F, Y293F, and Y292F in LTK cells yielded identical results. These data indicate that only two of the 6 conserved helical tyrosines residues, located in helices 4 and 7, are essential for full activity, but not for IAPS‐forskolin binding of the GLUT4.
Molecular cloning of the human type VIII adenylyl cyclase
Authors: Defer Nicole , Marinx Olivier , Stengel Dominique , Danisova Alena , Iourgenko Vadim , Matsuoka Isao , Caput Daniel , Hanoune Jacques
Published: July 2002

A cDNA coding for a human type VIII adenylyl cyclase has been isolated from human newborn brain‐stem tissue. This cDNA is 6,005 bp long and encodes for a protein of 1251 amino acids, exhibiting the two sets of six transmembrane spanning regions and the hydrophobicity profile typical of other mammalian adenylyl cyclases. Comparison with the rat form shows that they share 97% identity in amino acids. Type VIII adenylyl cyclase is unique in that it has both a long carboxy terminal and a long amino terminal tail. This is the first report on a complete cDNA clone coding for a human adenylyl cyclase. The distribution and regulation of this particular adenylyl cyclase suggest that it may be involved in learning, in memory and in drug dependence.
Specific thiol compounds complement deficiency in c‐type cytochrome biogenesis in Escherichia coli carrying a mutation in a membrane‐bound disulphide isomerase‐like protein
Authors: Sambongi Yoshihiro , Ferguson Stuart J.
Published: July 2002

Escherichia coli JCB606 carries a mutation in the dipZ gene, known to code for a disulphide isomerase‐like protein, with the consequence that holo forms of neither exogenous nor endogenous c‐type cytochromes are synthesised. This failure has been overcome by adding compounds containing thiol groups to the growth medium. Only l‐Cysteine and 2‐mercaptoethane sulphonic acid were effective, suggesting a (stereo)specific binding site that could be occupied by these compounds in the absence of the catalytic domain of DipZ.
The oxygenase reaction of acetolactate synthase detected by chemiluminescence
Authors: Durner Jörg , Gailus Valérie , Böger Peter
Published: July 2002

In addition to the synthesis of ketolacids the enzyme acetolactate synthase shows an oxygen‐consuming side reaction. Partially purified acctolactate synthase from corn (Zea mays L.) and barley (Hordeum vulgare L.) exhibits chemiluminescence in the presence of oxygen, Mn2+ and low concentrations of pyruvate. Light emission is inhibited by azide, but not by catalase or superoxide dismutase. The data suggest the formation of singlet oxygen during the catalytic cycle, and provides a basis for a highly sensitive assay for the oxygenase reaction of acetolactate synthase. Both synthase activity and chemiluminescence are inhibited by sulfonylurea herbicides. The results add a new aspect to the irreversible inhibition of acetolactate synthase by these herbicides which may be enhanced by the presence of reactive oxygen species.
Differentiation of PC12 cells in response to a cAMP analogue is accompanied by sustained activation of mitogen‐activated protein kinase
Authors: Young Stephen W. , Dickens Martin , Tavaré Jeremy M.
Published: July 2002

It has been proposed previously that the sustained activation of mitogen‐activated protein kinase may be necessary for the differentiation of PC12 cells. Differentiation of PC12 cells is induced by many extracellular agonists including nerve growth factor (NGF) and cyclicAMP analogues, but not epidermal growth factor (EGF), insulin or phorbol esters. Our results demonstrate that: (i) 8‐(4‐chlorophenylthio)‐cyclicAMP (CPT‐cAMP) activates MAP kinase; this raises the possibility that the MAP kinase pathway may be activated by agents that act through adenylate cyclase; (ii) NGF and CPT‐cAMP as well as phorbol esters promote sustained activation of MAP kinase. This suggests that while sustained MAP kinase activation may be associated with differentiation it may not be sufficient, and that other as yet unidentified parallel pathways may be involved.
Unique cleavage specificity of ‘prohormone thiol protease’ related to proenkephalin processing
Authors: Azaryan Anahit V. , Hook Vivian Y.H.
Published: July 2002

‘Prohormone thiol protease’ (PTP) represents the major enkephalin precursor processing activity in chromaffin granules. In this study, cleavage specificity of PTP for paired basic and monobasic residues was examined with a series of model peptide‐MCA (‐methylcoumarinamide) substrates. Monobasic peptides were cleaved at the COOH‐ and NH2‐terminal sides of the single basic residue. Dibasic peptides, however, were preferentially cleaved at the NH2‐terminal side of the pair, or between the two basic residues, with low cleavage at the COOH‐terminal side of the pair. Inhibition by the peptide inhibitor (d‐Tyr)‐Glu‐Phe‐Lys‐Arg‐CH2Cl provided further evidence for ptp's specificity for the dibasic Lys‐Arg site. Inhibition by Z‐Leu‐Val‐Gly‐CHN2; and Z‐Arg‐Leu‐Val‐Gly‐CHN2; suggests involvement of Val‐Gly in substrate binding to PTP; these two cystatin C‐related inhibitors also indicate PTP as a cysteine protease. These results demonstrate PTP's unique cleavage specificity that differs from other processing endopeptidases, including the subtilisin‐related proprotein convertases, PC1/PC3, and PC2, as well as the pituitary proopiomelanocortin‐converting enzyme, PCE. This study provides further evidence for PTP as a novel prohormone processing enzyme that belongs to the class of cysteine proteases.
Stimulation of cAMP accumulation by the cloned Xenopus melatonin receptor through Gi and Gz proteins
Authors: Yung Lisa Y. , Tsim Siu-Tai , Wong Yung H.
Published: July 2002

The Xenopus melatonin receptor was expressed in human embryonic kidney 293 cells and assayed for cAMP accumulation. In transfected 293 cells expressing the melatonin receptor, melatonin dose‐dependently inhibited the endogenous adenylyl cyclases. In contrast, melatonin stimulated the accumulation of cAMP in cells co‐expressing the type II adenylyl cyclase. Both the inhibitory and stimulatory responses to melatonin were mediated via Gi‐like proteins as they were blocked by pertussis toxin. Upon co‐transfection with the α subunit of Gz, the ability of melatonin to regulate both type II and the endogenous adenylyl cyclases became refractory to pertussis toxin, indicating that the melatonin receptor can also couple to Gz. However, other pertussis toxin‐insensitive G proteins such as Gq, G12 and G13 were unable to interact with the melatonin receptor.
Enzymatic characterization of purified NS3 serine proteinase of hepatitis C virus expressed in Escherichia coli
Authors: Moria Akiko , Yamada Kazunori , Kimura Junko , Koide Tomoko , Yuasa Satoshi , Yamadaa Ei , Miyamura Tatsuo
Published: July 2002

Non‐structural protein 3 (NS3) of the hepatitis C virus (HCV) has been shown to be a serine proteinase which cleaves the HCV polyprotein thus activating its replicative machinery. To characterize enzymatic activities of NS3 serine proteinase, the proteinase region was expressed in Escherichia coli and purified. The purified proteinase specifically cleaved a purified fusion protein sandwiching the NS5A/5B cleavage sequence. In addition to serine proteinase inhibitors, some chelators also inhibited the cleavage activity. Metal ions were not required for its activity, suggesting that the proteinase may be a novel serine proteinase having a unique binding site for chelators.
Identification of a novel secretory leukocyte protease inhibitor‐binding protein involved in membrane phospholipid movement
Authors: Tseng Ching-Chung , Tseng Ching-Ping
Published: July 2002

Previous studies have suggested that human salivary secretory leukocyte protease inhibitor (SLPI) inhibits HIV‐1 by binding to a host cell surface protein of unknown identity. Using the yeast two‐hybrid assay, we identified a gene sequence encoding a novel SLPI‐binding protein (SLPI‐BP). The 1.5‐kb cDNA encodes a 318‐amino acid protein with a predicted transmembrane segment near the C‐terminus. Sequence analysis revealed that SLPI‐BP is the human scramblase protein that is involved in the movement of membrane phospholipids. Co‐expression of SLPI and SLPI‐BP followed by an S‐protein pulldown assay confirmed the specific interaction between these two proteins. Our data represent the first report for the identity of SLPI‐BP.
Solution NMR structures of the polyene macrolide antibiotic filipin III
Authors: Volpon Laurent , Lancelin Jean-Marc
Published: July 2002

The solution structure of filipin III, an antifungal polyene macrolide biosynthesized by Streptomyces filipinensis and widely used for the detection and the quantitation of cholesterol in biomembranes, has been calculated with a set of geometrical restraints derived from 1H NMR in DMSO‐d
6 at 25°C. Filipin III appears as a rod‐shaped molecule of 18 Å length. Its amphiphilic structure is made of an all‐syn 1,3‐polyol motif, stabilized by intramolecular hydrogen bonds on one side, and a conjugated pentaene moiety on the other side of the molecule. The overall shape is comparable to cholesterol, and the molecular structure of filipin III affords a first molecular basis to the comprehensive understanding of the interactions possible in the filipin III–cholesterol complex which is still unknown at the atomic resolution.
Inhibition of inducible nitric oxide synthesis by oxidized lipoprotein(a) in a murine macrophage cell line
Authors: Moeslinger Thomas , Friedl Roswitha , Volf Ivo , Brunner Monika , Koller Elisabeth , Spieckermann Paul Gerhard
Published: July 2002

Increased plasma levels of human lipoprotein(a) (Lp(a)) are highly correlated with the development of atherosclerotic lesions. During our study, we investigated the effects of native and hypochlorite oxidized lipoprotein(a) (ox‐Lp(a)) on nitric oxide production by the inducible nitric oxide synthase (iNOS) in lipopolysaccharide/interferon stimulated mouse macrophages (J774A.1). Ox‐Lp(a) (0–2 μg/ml) induces a dose dependent inhibition of inducible nitric oxide synthesis. iNOS protein expression showed a dose dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of ox‐Lp(a). Ox‐Lp(a) decreases iNOS mRNA synthesis as shown by reverse transcription‐polymerase chain reaction. Ox‐Lp(a) induced iNOS inhibition might contribute to the development of atherosclerotic lesions by reducing the anti‐atherogenic effects of nitric oxide.
A novel type of FKBP in the secretory pathway ofNeurospora crassa
Authors: Solscheid Bernd , Tropschug Maximilian
Published: July 2002

FKBPs define a subfamily of peptidyl‐prolylcis/transisomerases (PPIases). PPIases are known to play roles in cellular protein folding, protein interactions and signal transduction. Here we describe NcFKBP22 fromNeurospora crassa, a novel type of FKBP. NcFKBP22 is synthesized as a precursor protein with a cleavable signal sequence. In addition to a typical FKBP domain in the amino‐terminal part mature NcFKBP22 contains a novel second domain which is unique amongst all known FKBPs. The amino acid composition of this carboxy‐terminal domain is highly biased. Secondary structure predictions suggest that this domain may form an amphipathic α‐helix. The carboxy‐terminus of NcFKBP22 is –HNEL, a potential endoplasmic reticulum (ER) retention signal, suggesting that NcFKBP22 is a resident protein of the ER.
Long‐chain fatty acids promote opening of the reconstituted mitochondrial permeability transition pore
Authors: Wiȩckowski Mariusz R. , Brdiczka Dieter , Wojtczak Lech
Published: July 2002

Adenine nucleotide translocase–porin–hexokinase complex isolated from rat brain, when reconstituted into phospholipid‐cholesterol vesicles, exhibits all properties of the mitochondrial permeability transition pore [Beutner, G., Rück, A., Riede, B., Welte, W. and Brdiczka, D. (1996) FEBS Lett. 396, 189–195]. In the present work, the effect of long‐chain fatty acids on such reconstituted pore was examined. Opening of the pore was measured by leakage of either malate or fluorescein sulphonate entrapped inside the vesicles. It was found that myristate and oleate in the presence of 50 or 100 μM Ca2+ produced a partial release of the probes in a dose‐dependent way. A dicarboxylic fatty acid analogue, that appeared inactive as protonophore in intact mitochondria, exerted no effect on pore opening in the reconstituted system. 100 μM Ca2+ alone was without effect. Pore opening by fatty acids in the reconstituted system was partly prevented by cyclosporin A. The pore opening also occurred when the vesicles were incubated in the presence of pancreatic phospholipase A2. In this case, the opening was decreased by cyclosporin A or serum albumin. These results indicate that long‐chain fatty acids elicit opening of the permeability transition pore reconstituted in phospholipid vesicles in a similar way as in intact mitochondria [Wiȩckowski, M.R. and Wojtczak, L. (1998) FEBS Lett. 423, 339–342].