bluuebunny/crossref_metadata_2025 huggingface dataset:
A major baker's asthma allergen from rye flour is considerably more active than its barley counterpart
Authors: García-Casado Gloria , Armentia Alicia , Sánchez-Monge Rosa , Sánchez Luis M. , Lopez-Otín Carlos , Salcedo Gabriel
Published: April 2003

A rye flour protein of about 13.5 kDa, as well as its barley homologue, have been isolated. The rye component was recognized in vitro by IgE of allergic patients and provoked positive responses in 15 out of 21 baker's asthma patients (71%) when skin prick tests were performed. Its barley homologue showed no detectable in vitro reactivity and caused positive responses in only one‐third of patients. Although no inhibitory activity against different α‐amylases or trypsin was found for these two proteins, their N‐terminal sequencing revealed considerable similarity to several members of the cereal α‐amylase/trypsin inhibitor family.
Effects of protein kinase modulators on transferrin receptor expression in human leukaemic HL‐60 cells
Authors: Lok C.N. , Chan K.-F.J. , Loh T.T.
Published: April 2003

The mRNA of transferrin receptor (TfR) is constitutively expressed in proliferating human leukaemic HL‐60 cells. Treatment of HL‐60 cells with phorbol 12‐myristate 13‐acetate (PMA), a protein kinase C (PKC) activator, or dibutyryl‐cyclic AMP (dbcAMP), a protein kinase A (PKA) activator, resulted in a 90% decrease in the level of TfR mRNA. Inhibition of TfR mRNA expression induced by 10 nM PMA and 100 μM dbcAMP was abolished by prior incubation of cells with 0.1–1.0 μM GF109203X, a PKC‐specific inhibitor, and 1–10 μM H‐89, a PKA‐specific inhibitor, respectively. The blocking effects of GF109203X and H‐89 were dose‐dependent and complete at the highest concentrations of the inhibitors used. Although treatment of cells with GF109203X or H‐89 alone did not alter the constitutive expression of TfR mRNA, incubation of cells with 30–100 nM staurosporine, a wide‐spectrum protein kinase inhibitor, resulted in suppression of the constitutive expression of TfR mRNA in a dose‐dependent manner. These results suggest that (i) the down‐regulation of TfR mRNA expression during the differentiation of HL‐60 cells can be mediated by activation of either PKC or PKA; (ii) the constitutive expression of TfR mRNA in proliferating HL‐60 cells is staurosporine‐sensitive and is probably maintained by protein kinase(s) other than PKC and PKA.
Boar spermadhesin PSP‐II: Location of posttranslational modifications, heterodimer formation with PSP‐I glycoforms and effect of dimerization on the ligand‐binding capabilities of the subunits
Authors: Calvete Juan José , Mann Karlheinz , Schäfer Wolfram , Raida Manfred , Sanz Libia , Töpfer-Petersen Edda
Published: April 2003

Spermadhesin PSP‐II was isolated from the non‐heparin‐binding fraction of boar seminal plasma; its disulphide bridge pattern, and the location of a single N‐glycosylation site were established. PSP‐II forms a heterodimer with specific N‐glycoforms of PSP‐I. Although both subunits possess heparin‐binding capability, the PSP‐I/PSP‐II complex does not. The heterodimer contains binding sites for zona pellucida glycoproteins and soybean trypsin inhibitor located in the PSP‐II subunit. However, the PSP‐I/PSP‐II heterodimer binds only loosely to the sperm surface and is easily removed during in vitro capacitation, suggesting that the zona pellucida binding activity may not be relevant for gamete interaction. Our results show that dimerization of spermadhesins PSP‐I and PSP‐II markedly affects their binding capabilities.
Effect of a negative regulatory element (NRE) on the human CYP1A1 gene expression in breast carcinoma MCF‐7 and hepatoma HepG2 cells
Authors: Jørgensen Eva Cecilie Bonefeld , Autrup Herman
Published: April 2003

The expression of the cytochrome P4501A1 gene, CYP1A1, is induced by e.g. 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) mainly by transcriptional mechanisms. The inducers mediate their effect upon binding and activation of the aryl hydrocarbon receptor (AHR) transcription‐factor complex. Utilizing chimeric CYP1A1/HCAT constructs transient gene expression experiments indicate that the putative negative regulatory element (NRE) of CYP1A1 influence the relative TCDD induced CAT activity in HepG2 cells, whereas this effect was not observed in MCF‐7 cells. Differences in the formation of cell‐specific protein‐DNA complexes were demonstrated by gel retardation assays suggesting a functional difference of NRE in these two cell lines.
Deletion mutagenesis of heparin cofactor II: defining the minimum size of a thrombin inhibiting serpin
Authors: Sheffield William P. , Blajchman Morris A.
Published: April 2003

Heparin cofactor II (HCII) is a 66 kDa plasma glycoprotein that belongs to the serpin superfamily of protease inhibitors. Its natural target is thrombin. HCII inhibits thrombin in both a progressive reaction, and in an accelerated reaction catalyzed by a glycosaminoglycan, dermatan sulphate (DS). Both modes of inhibition result in the formation of a stable, denaturation‐resistant complex. Using a cDNA clone encoding rabbit HCII recently isolated and characterized in our laboratory, we have employed deletion mutagenesis to identify amino‐terminal regions of the molecule which are essential to the progressive reaction. PCR was employed to produce four deletion constructs: Δ58, Δ81, Δ106, and Δ169, all in an in vitro transcription vector plasmid background. Transcription of the full‐length construct, and of the four deletion constructs, followed by in vitro translation in rabbit reticulocyte lysate, was used to produce the corresponding HCII‐related polypeptides. The Δ106 and Δ169 mutants failed to react with thrombin, even in the presence of DS. In contrast, the Δ58 and Δ82 mutants retained the ability to form complexes with thrombin, although the rate of complex formation was decreased for the latter mutant compared to the full‐length recombinant HCII; no acceleration of complex formation in the presence of 20 μg/ml DS was noted for either truncated recombinant HCII. Alignment of the rabbit HCII primary structure with secondary structural elements found in α
1antitrypsin and other serpins showed that the non‐functional Δ106 mutant lacks helix A, while the functional Δ82 mutant contains this element. Our results suggest that helix A is an essential part of a functional serpin, and define the limits of the amino‐terminal region of HCII which is not essential for thrombin inhibition.
Analysis and crystallization of a 25 kDa C‐terminal fragment of cloned elongation factor Ts fromEscherichia coli
Authors: Bøgestrand Søren , Wiborg Ove , Thirup Søren , Nyborg Jens
Published: April 2003

A 25 kDa C‐terminal tryptic fragment of elongation factor Ts has been purified to homogeneity. Experimental evidence suggests that the 25 kDa C‐terminal and the 5.3 kDa N‐terminal fragments are structurally independent domains. The N‐terminal fragment is shown to be essential for the nucleotide exchange activity. Crystals of the C‐terminal fragment belong to space group P2 or P21. The diffraction pattern shows a pronounced pseudo‐C2 symmetry at low resolution. This pseudo symmetry increases when the crystals are irradiated with X‐rays for a few hours.
Redox properties of the sulfhydrogenase from Pyrococcus furiosus
Authors: Arendsen Alexander F. , Veenhuizen Peter Th.M. , Hagen Wilfred R.
Published: April 2003

The sulfhydrogenase from the extreme thermophile Pyrococcus furiosus has been re‐investigated. The αβγδ heterotetrameric enzyme of 153.3 kDa was found to contain 17 Fe, 17 S2−, and 0.74 Ni. The specific activity of the purified protein was 80 U/mg. Three EPR signals were found. A rhombic signal (g = 2.07, 1.93, 1.89) was observed reminiscent in its shape and temperature dependence of spectra from [4Fe‐4S](2+;1+) clusters. However, in reductive titrations the spectrum appeared at the unusually high potential E
m,7.5 = −90 mV. Moreover, the signal dissappeared again at E
m,7.5 = −328 mV. Also, two other signals appear upon reduction: a near‐axial (g = 2.02, 1.95, 1.92) spectrum (E
m,7.5 = −303 mV) indicative for the presence of a [2Fe‐2S](2+;1+) cluster, and a broad spectrum of unknown origin with effective g‐values 2.25, 1.89 (E
m,7.5 = −310 mV). We hypothesize that the latter signal is caused by magnetic interaction of the rhombic signal and a third cluster.
Related organelles of the endosome‐lysosome system contain a different repertoire of ubiquitinated proteins in Sf9 insect cells
Authors: Löw Péter , Doherty Fergus J. , Fellinger Erzsébet , Sass Miklós , John Mayer R. , László Lajos
Published: April 2003

Two components of the endosomal/lysosomal compartment of Sf9 cells, multivesicular bodies (MVB) and light vacuoles with membrane complexes (LVMC) have been isolated and probed for ubiquitin protein conjugates with a specific antibody. Immunogold electron microscopy indicates that whereas ubiquitin‐protein conjugates are localised to electron dense areas of MVB they are associated with the membranes of LVMC. Five ubiquitinated polypeptides are revealed in MVB by immunoblotting while numerous ubiquitinated species forming a smear following electrophoresis are present in LVMC. We suggest two possible routes for entry of ubiquitin‐protein conjugates into these organelles, via the cell surface and via primary lysosomes.
Conformational stability of Cys45‐alkylated and hydrogen peroxide‐oxidised glutathione S‐transferase
Authors: Sluis-Cremer Nicolas , Dirr Heini
Published: April 2003

A highly reactive cysteine residue in class pi glutathione S‐transferases enhances their susceptibility to chemical alkylation and oxidative stress. Alkylation of the reactive Cys45 in the porcine class pi enzyme (pGSTP1‐1) with either N‐iodoacetyl‐N′‐(5‐sulpho‐1‐naphthyl)ethylenediamine or iodoacetamide results in a loss of enzyme activity and glutathione‐binding function. Similarly, oxidation of pGSTP1‐1 with hydrogen peroxide (H2O2) also results in a loss of catalytic and gluthathione‐binding function, but these effects are reversed by the addition of 5 mM glutathione or dithiothreitol. Analysis by SDS‐PAGE of the H2O2‐oxidised enzyme indicates oxidation‐induced formation of disulphide bonds involving Cys45. Equilibrium‐unfolding studies with guanidinium chloride indicate that the unfolding of Cys45‐alkylated and H2O2‐oxidised pGSTP1‐1 can be described by a two‐state model in which the predominant thermodynamically stable species are the folded dimer and unfolded monomer. Unfolding transition curves suggest that the introduction of a large and bulky AEDANS at Cys45 does not affect the unfolding pathway for pGSTP1‐1. H2O2‐oxidised pGSTP1‐1, on the other hand, appears to follow a different unfolding pathway. This appears not to be a result of the introduction of disulphide bonds since the reduction of these bonds in the oxidised protein with dithiothreitol does not affect the unfolding transition. Furthermore, the conformational stability of the oxidised protein is significantly diminished (ΔG(H2O) = 11.6 kcal/mol) when compared with unmodified and AEDANS‐alkylated enzyme (ΔG(H2O) = 22.5 kcal/mol).
Interaction of smooth muscle calponin with phospholipids
Authors: Bogatcheva Natalia V. , Gusev Nikolai B.
Published: April 2003

Analyzing the primary structure we predicted that calponin may interact with phospholipids. In order to check this suggestion we investigated the interaction of calponin with phospholipids by ultracentrifugation, light scattering, vesicles leakage and differential scanning calorimetry. In agreement with our prediction calponin interacts with acidic phospholipids and the phospholipid‐binding site was located in the short (13 kDa) N‐terminal chymotryptic peptide of calponin. The apparent dissociation constant of calponin‐phospholipids complex was less than 0.2 μM and calmodulin competes with phospholipids for calponin binding. Although the interaction of calponin with phospholipids decreases at high ionic strength, calponin binds phospholipids even in the presence of 100–150 mM of the salt. Under certain conditions calponin induced leakage of phospholipid vesicles and affected the cooperativity of lipid phase transition. It is concluded that both electrostatic and hydrophobic interactions provide for calponin‐phospholipid complex formation.
Oxidation reactions catalyzed by manganese peroxidase isoenzymes from Ceriporiopsis subvermispora
Authors: Urzúa Ulises , Larrondo Luis Fernando , Lobos Sergio , Larraín Juan , Vicuña Rafael
Published: April 2003

A total of 11 manganese peroxidase isoenzymes (MnP1‐MnP11) with isoelectric points (pIs) in the range of 4.58–3.20 were isolated from liquid‐ and solid‐state cultures of the basidiomycete Ceriporiopsis subvermispora. In the presence of hydrogen peroxide, these isoenzymes showed different requirements for Mn(II) in the oxidation of vanillylacetone, o‐dianisidine, p‐anisidine and ABTS, whereas oxidation of guaiacol by any isoenzyme did not take place when this metal was omitted. K
m values for o‐dianisidine and p‐anisidine in the absence of Mn(II) are in the range of 0.5–1.0 mM and 4.5–42.0 mM, respectively. Oxalate and citrate, but not tartrate, accelerate the oxidation of o‐dianisidine, both in the presence and in the absence of Mn(II). MnPs from this fungus are able to oxidize kojic acid without externally added hydrogen peroxide, indicating that they can also act as oxidases. In this reaction, however, the requirement for Mn(II) is absolute.
The lipoxygenase pathway in garlic (Allium sativum L.) bulbs: detection of the novel divinyl ether oxylipins
Authors: Grechkin Alexander N. , Fazliev Farit N. , Mukhtarova Lucia S.
Published: April 2003

Incubations of [1‐14C]linoleic acid or [1‐14C]‐(9Z.11E, 13S)‐13‐hydropero xy‐9,11‐octadecadienoic acid (13‐HPOD) with juice of garlic bulbs lead to the formation of one predominant labelled product, viz., the novel divinyl ethery (9Z,11E, 1′E)‐12‐(1′‐hexenyloxy)‐9,11‐odecadienoic acid (‘etheroleic acid’). With lesser efficiency [1‐14C]α‐linolenic acid or [1‐14C](9Z,11E,13S,15Z)‐13‐hydroperoxy‐9,11,15‐octadecatrienoic acid (13‐HPOT) are converted in this way into (9Z,11E,1′E,3′Z)‐12‐(1′,3′‐hexadienyloxy)‐9,11‐dodecadienoic acid (‘etherolenic acid’). Thus, garlic bulbs possess the activity of a new 13‐hydroperoxide‐specific divinyl ether synthase.
β‐Sheet secondary structure of an LDL receptor domain from complement factor I by consensus structure predictions and spectroscopy
Authors: Ullman Christopher G. , Haris Parvez I. , Smith Kathryn F. , Sim Robert B. , Emery Vincent C. , Perkins Stephen J.
Published: April 2003

Low density lipoprotein receptor domains (LDLrs) represent a large cell surface receptor superfamily of consensus length 39 residues. Alignment of 194 sequences indicated highly conserved Cys and Asp/Glu residues, and a consensus secondary structure with three β‐strands was predicted. Sequence threading against known protein folds indicated consistency with small β‐sheet proteins. Complement factor I contains two LDLrs, and the second of these was successfully expressed using a bacterial pGEX system. FT‐IR spectroscopy on this indicated a small amount of β‐sheet together with turns and loops. LDLr is proposed to have a β‐sheet structure in which the five biologically important Asp/Glu residues are located on an exposed loop.
1H‐NMR and photo‐CIDNP spectroscopies show a possible role for Trp23 and Phe31 in nucleic acid binding by P2 ribonuclease from the archaeon Sulfolobus solfataricus
Authors: Consonni Roberto , Limiroli Rita , Molinari Henriette , Fusi Paola , Grisa Margareth , Vanoni Marco , Tortora Paolo
Published: April 2003

Investigations were performed on recombinant ribonuclease P2 from Sulfolobus solfataricus, previously cloned and expressed in Escherichia coli [Fusi, P., Grisa, M., Mombelli, E., Consonni, R., Tortora, P. and Vanoni, M. (1995) Gene 154, 99–103]. NMR and photo‐CIDNP spectroscopies showed that the enzyme possesses an aromatic cluster constisting of Phe5, Tyr7, Phe31 and Tyr33 while Trp23 is fully exposed to solvent. Phe31, Tyr33 and Trp23 are located within a triple stranded antiparallel β‐sheet, each one being part of an amino acid stretch matching consensus sequences for RNA binding. Phe31 and Trp23 are exposed to and specifically interact with a flavin dye used as a model ligand, with a topology reminiscent of that found in several eubacterial and eukariotic RNA‐binding proteins.
Analogue versus digital recognition of DNA by bleomycin: an effect of the carbohydrate moiety
Authors: Bailly Christian , Kénani Abderraouf , Waring Michael J.
Published: April 2003

We have sought to determine the influence of the carbohydrate moiety of the antitumour antibiotic bleomycin on the sequence‐specific cleavage of DNA. Both bleomycin A2 and deglycobleomycin A2 produce different cleavage patterns with DNA in which the 2‐amino group has been removed from guanine, added to adenine, or both, as well as on a designed DNA fragment containing a few defined cleavage sites. Although each drug cleaves DNA primarily at Gp and Gp sites, the cleavage at these sites is frequently found to be stronger with deglycobleomycin compared with bleomycin A2. Conversely, in most cases the cleavage at secondary sites, in particular at ApT steps, is significantly reduced or even abolished with deglycobleomycin. The results indicate that the gulose‐mannose moiety of bleomycin A2 plays a significant role in the recognition of preferred nucleotide sequences and confirm the view that both secondary structure and interaction with guanine are involved in determining sequence‐specific cleavage of DNA by bleomycin.
Silencing of the epidermal growth factor receptor in the absence of the ligand requires phospholipase C activity
Authors: Langgut Werner , Ogilvie Adaling
Published: April 2003

The possible involvement of phospholipase Cβ (PLCβ) in a crosstalk mechanism between G‐protein coupled receptors and receptor tyrosine kinases was investigated in HeLa‐S3 and A‐431 cells. A basic activity of the receptor for epidermal growth factor (EGF) in the absence of its ligand was found only in A‐431 cells overexpressing this receptor. Inhibition of PLC drastically increased EGF receptor activity in both cell lines, suggesting that PLC activity is necessary for the silencing of the EGF receptor in the absence of its ligand. Activation of PLCβ and protein kinase C (PKC) via G‐protein‐linked ATP receptors greatly diminished the basic EGF receptor activity in A‐431 cells. This negative regulation was prevented by the protein tyrosine phosphatase inhibitor, vanadate. The results suggest a crosstalk between a G‐protein‐linked receptor and a receptor tyrosine kinase, involving signalling via PLCβ and PKC to a downstream protein tyrosine phosphatase functioning in the control of EGF receptor activity.
Identification of the primary growth response gene, ST2/T1, as a gene whose expression is differentially regulated by different protein kinase C isozymes
Authors: Kieser Arnd , Goodnight JoAnne , Kölch Walter , Mischak Harald , Frederic Mushinski J.
Published: April 2003

Individual protein kinase C isozymes have been shown to play different roles in mediating proliferation, differentiation and transformation, but it is not known to what extent these effects involve induction of expression of particular genes. To explore the differential gene expression that might be induced by activation of different PKC isozymes, we stably transfected NIH 3T3 cells with expression vectors that encode the isozymes PKC‐α, ‐βII, ‐γ, ‐δ, ‐ϵ, ‐ζ and ‐η. Using differential display‐reverse transcription‐polymerase chain reaction we isolated a small cDNA that encodes a portion of the primary response gene, ST2 (also referred to as T1 or DER4), and we confirmed by RNA blot studies that ST2/T1 expression is differentially regulated by PKC isozymes. ST2/T1 mRNA is undetectable in the unstimulated parental NIH 3T3 cells that express only the α isozyme of PKC, but it can be induced by phorbol ester treatment. Clones that overexpress PKC‐α, ‐δ or ‐ϵ similarly do not express ST2/T1 until they are stimulated with phorbol esters, which induces expression of ST2/T1 with kinetics similar to wild‐type NIH 3T3 but to different extents. In contrast, ST2/T1 mRNA is already present in unstimulated cells that overexpress PKC‐βII, ‐γ, ‐ζ and ‐η, but phorbol ester greatly enhances ST2/T1 expression in these cells. These results suggest a differential role for PKC isozymes in mediating the ST2/T1 expression that is induced by growth stimuli.
Effect of the PufQ protein on early steps in the pathway of bacteriochlorophyll biosynthesis in Rhodobacter capsulatus
Authors: Fidai Shafique , Dahl Jeffery A. , Richards William R.
Published: April 2003

The addition in trans of the pufQ gene to a strain of Rhodobacter capsulatus from which the entire puf operon had been deleted, increased its ability to synthesize coproporphyrinogen from both δ‐aminolevulinic acid and porphobilinogen. Studies at the enzyme level indicated that the conversion of porphobilinogen to uroporphyrinogen III had about a 2‐fold higher level of activity in the anaerobically‐grown pufQ‐containing strain. This increase in activity over the puf‐deletion strain appeared to occur during transitions from aerobic to semiaerobic growth conditions. These results indicated that the PufQ protein may exert a stimulatory effect quite early in the pathway of bacteriochlorophyll biosynthesis.
[3H]9‐Methyl‐7‐bromoeudistomin D, a caffeine‐like powerful Ca2+ releaser, binds to caffeine‐binding sites distinct from the ryanodine receptors in brain microsomes
Authors: Yoshikawa Kazumori , Furukawa Ken-Ichi , Yamamoto Masayuki , Momose Kazutaka , Ohizumi Yasushi
Published: April 2003

[3H]9‐Methyl‐7‐bromoeudistomin D ([3H]MBED), the most powerful Ca2+ releaser from sarcoplasmic reticulum, specifically bound to the brain microsomes. Caffeine competitively inhibited [3H]MBED binding. [3H]MBED binding was markedly blocked by procaine, whereas that was enhanced by adenosine‐5′‐(β,γ‐methylene)triphosphate. The B
max value was 170 times more than that of [3H]ryanodine binding. The profile of sucrose‐density gradient centrifugation of solubilized microsomes indicated that [3H]MBED binding protein was different from [3H]ryanodine binding protein. These results suggest that there are MBED/caffeine‐binding sites in brain that are distinct from the ryanodine receptor and that MBED becomes an essential molecular probe for characterizing caffeine‐binding protein in the central nervous system.
Purification from human plasma of a tetrapeptide that potentiates insulin‐like growth factor‐I activity in chick embryo cartilage
Authors: Straczek J. , Maachi F. , Le Nguyen D. , Becchi M. , Heulin M.H. , Nabet P. , Belleville F.
Published: April 2003

Human plasma has been shown to contain a low molecular weight factor that potentiates human IGF‐I stimulation of glycosaminoglycan synthesis in chick embryo cartilage. The peptide was purified and characterized by Edman degradation and electrospray mass spectrometry. The primary structure determined was: Trp‐Gly‐His‐Glu. A homologous synthetic peptide similarly promoted matrix biosynthesis in cartilage exposed to IGF‐1.
Two identical hydrophobic clusters are present on the same actin monomer: interaction between one myosin subfragment‐1 and two actin monomers
Authors: Labbé Jean-Pierre , Boyer Mireille , Benyamin Yves
Published: April 2003

Two‐dimensional hydrophobic clusters analysis (HCA) was used to compare the distribution of hydrophobic clusters along various actin sequence. HCA‐deduced patterns were not altered by amino‐acid variations throughout the evolution of actin and we observed similar hydrophobic motifs comprising myosin subfragment‐1 ATP‐independent binding sites. HCA suggested the presence of two groups of identical hydrophobic motifs (A1 and A2) which bound on each side of the S1 (63 kDa‐31 kDa) connecting segment in relation with two actin monomers. This connection is important in communications between actin‐ and nucleotide‐binding sites. We postulate that some relation and message between the two motifs A1 and A2 take place through myosin subfragment‐1 (63 kDa‐31 kDa) connecting segment.
Chemical cross‐linking leads to two high molecular mass aggregates of rat α1β1 integrin differing in their conformation but not in their composition
Authors: Löster Klemens , Baum Oliver , Hofmann Werner , Reutter Werner
Published: April 2003

In order to detect protein interactions of the collagen/laminin receptor α
1
β
1 integrin, covalent chemical cross‐linking was performed with the homo‐bifunctional, amine reactive reagents DSS (disuccinimidylsuberate) and DSP (dithiobis(succinimidylpropionate)). After cross‐linking of the 190 kDa rat α
1 integrin subunit, immunoblotting revealed two additional, immunoreactive, high molecular mass complexes (M
r 240/290 k). Generation of the 240/290 kDa aggregates depended on the presence of the intact tertiary protein structure. As shown with immunoaffinity purified proteins, the 240/290 kDa aggregates consist exclusively of α
1 and β
1 integrin subunits. No other cross‐linked proteins associated with the α
1 or β
1 subunit were detected. In contrast to the non‐cross‐linkable α
1
β
1 integrin, the 240/290 kDa aggregates presumably represent active forms of the adhesion receptor, because both bound in vitro to collagen I and IV. This ability of α
1
β
1 integrin to cross‐link and produce two additional high molecular mass forms is shared by rat α
9
β
1 integrin. Thus, the cross‐linking approach directly indicates that β
1 integrins occur in different conformations caused by variations in the folding and/or spatial arrangement of their subunits.
Principles of symmetrical organization for the pyruvate dehydrogenase complex
Authors: Goldstein Boris N. , Saifullin Salavat R. , Zakrzhevskaya Dina T.
Published: April 2003

The experimentally observed phenomenon of non‐equimolarity for enzyme components, assembled into multienzyme complexes of the 2‐oxo acid dehydrogenases family, is structurally interpreted to predict the only possible stable symmetrical distribution of peripheral components on the complex core. To obey the equivalent neighboring, that is necessary for unique self‐assembled structures, we should deduce discrete conformational states for core subunits, those with different affinity for peripheral components. Two kinetically different types of substrate‐intermediate pathways through the lipoyl network of the mammalian pyruvate dehydrogenase complex follow from this structural theory. The theory predicts unusual kinetic behavior for the multienzyme complex.
Expression and refolding of a high‐affinity receptor binding domain from rat α1‐macroglobulin
Authors: Nielsen Kåre L. , Sottrup-Jensen Lars , Fey Georg H. , Thøgersen Hans C.
Published: April 2003

A recombinant version of the receptor binding domain of rat α
1‐macroglobulin (RBDv) consisting of residues 1319–1474 has been expressed in E. coli. Competition experiments with 125I‐labelled methylamine treated human α
2‐macroglobulin reveal that the α
1‐macroglobulin‐RBDv exhibit the same high affinity or the α
2‐acroglobulin receptor as the entire 40 kDa light chain from rat α
1‐macroglobulin. It is therefore concluded, that all determinants for receptor interaction reside in the C‐terminal approx. 150 residues of the α‐macroglobulin subunit.
NADPH‐sulfite reductase flavoprotein from Escherichia coli: Contribution to the flavin content and subunit interaction
Authors: Eschenbrenner Michel , Covès Jacques , Fontecave Marc
Published: April 2003

The flavoprotein component (SiR‐FP) of the sulfite reductase of E. coli is an octamer of the 66 kDa α subunit. It was shown to be cleaved in two peptide fragments. The 23 kDa fragment has been purified as a polymer of 8–10 subunits. It corresponds to the N‐terminal part of the native protein and was shown to contain essentially FMN as cofactor. The 43 kDa fragment is monomeric. It contains exclusively FAD and remains able to catalyze efficiently NADPH‐dependent reductions. One can conclude that each α‐chain of SiR‐FP is composed of two distinct domains, one binding FAD and the other FMN and that the FMN‐binding domains cooperate for a head‐to‐head subunit interaction.
Reduction of the tyrosyl radical and the iron center in protein R2 of ribonucleotide reductase from mouse, herpes simplex virus and E. coli by p‐alkoxyphenols
Authors: Pötsch Stephan , Sahlin Margareta , Langelier Yves , Gräslund Astrid , Lassmann Günter
Published: April 2003

The rate of reduction of the tyrosyl radical in the small subunit of ribonucleotide reductase (protein R2) from E. coli, mouse, and herpes simplex virus (HSV‐2) by a series of p‐alkoxyphenols with different alkyl chains, have been studied by stopped‐flow UV‐vis and stopped‐flow EPR spectroscopy. The reduction and release of iron in R2 by the inhibitors was followed using bathophenanthroline as chelator of Fe2+. p‐Alkoxyphenols reduce the mouse R2 tyrosyl radical 1–2 orders of magnitude faster than the HSV‐2 and E. coli radical. In contrast to E. coli, the iron center in R2 from mouse and HSV‐2 is reduced by the inhibitors. For mouse R2, the rate of reduction of the tyrosyl radical increases in parallel with increasing alkyl chain length of the inhibitor, an observation which may be important for the design of new antiproliferative drugs.
The complete Consensus V3 loop peptide of the envelope protein gp120 of HIV‐1 shows pronounced helical character in solution
Authors: Vranken Wim F. , Budesinsky Milos , Fant Franky , Boulez Kris , Borremans Frans A.M.
Published: April 2003

The disulfide bridge closed cyclic peptide corresponding to the whole Consensus V3 loop of the envelope protein gp120 of HIV‐1 was examined by proton 2D‐NMR spectroscopy in water and in a 20% trifluoroethanol/water solution. In water, NOE data support a β‐turn conformation for the central conservative GPGR region and point towards partial formation of a helix in the C‐terminal part. Upon addition of trifluoroethanol, a C‐terminal helix is formed. This is evidenced by NOE data, α‐proton chemical shift changes and changes in the J
Nα
vicinal coupling constants. The C‐terminal helix is amphipathic and also occurs in other examined strains. It could therefore be an important feature for the functioning of the V3 loop.
Expression of poliovirus 2Apro in mammalian cells: effects on translation
Authors: Aldabe Rafael , Feduchi Elena , Novoa Isabel , Carrasco Luis
Published: April 2003

Poliovirus protease 2Apro has been efficiently expressed in HeLa and COS cells upon transfection with vector pTM1‐2A and infection with the recombinant vaccinia virus bearing the T7 RNA polymerase. The expressed poliovirus protease localizes to the cytoplasm of the transfected cells, both in the endoplasmic reticulum and in vesicles scattered in the cytoplasm. Cleavage of p220, a component of initiation factor eIF‐4F, selectively occurs from 5 h post‐infection in transfected cells infected with the recombinant virus. This cleavage correlates in time with the profound inhibition observed in the synthesis of vaccinia virus proteins. A similar blockade of vesicular stomatitis virus translation takes place upon 2Apro expression. Finally, the synthesis of poliovirus protein 2C from a recombinant vaccinia virus that expresses this protein under the EMC untranslated leader region is not affected by the synthesis of 2Apro. These findings lend support to the idea that translation of capped mRNAs requires the integrity of p220, while this requirement is not observed when translation of a mRNA bearing a picornavirus leader region is assayed.
ClC‐6 and ClC‐7 are two novel broadly expressed members of the CLC chloride channel family
Authors: Brandt Silke , Jentsch Thomas J.
Published: April 2003

We cloned two novel members of the CLC chloride channel family from rat and human brain. ClC‐6 is a 97‐kDa protein, and ClC‐7 a 89‐kDa protein roughly 45% identical with ClC‐6. Together they define a new branch of this gene family. Both genes are very broadly expressed, e.g. in brain, testes, muscle and kidney. In mouse embryos, both genes are expressed as early as day 7. Wyile the human gene for ClC‐6 is located on human chromosome 1p36 and shares this region with hClC‐Ka and hClC‐Kb, ClC‐7 is on 16p13. ClC‐6 has a highly conserved glycosylation site between transmembrane domains D8 and D9, while ClC‐7 is the only known eukaryotic ClC protein which lacks this site. Hydropathy analysis indicates that domain D4 cannot serve as a transmembrane domain. Both ClC‐6 and ClC‐7 cannot be expressed as chloride channels in Xenopus oocytes, either singly or in combination.
Reversible denaturation of cyclosporin synthetase by urea
Authors: Dittmann Joachim , Vaillant François , Kleinkauf Horst , Lawen Alfons
Published: April 2003

The reversible denaturation of the multifunctional polypeptide, cyclosporin synthetase, by urea was analyzed. It is possible to discriminate between at least two stages of enzyme denaturation. While at low urea concentration (up to 0.8 M) cyclosporin A formation is inhibited, synthesis of the diketopiperazine cyclo‐(), a molecule representing a partial sequence of cyclosporin A is still detectable. At higher concentrations of urea the enzyme preparation is totally inactive. This inactivation is a consequence of conformational change(s) of cyclosporin synthetase as shown by fluorescence emission spectra of native and denatured enzyme. These data imply a consecutive folding/defolding mechanism for the different domains forming the multifunctional polypeptide.
Ionic transport in the plasma membrane of carrot protoplasts from embryogenic cell‐suspension cultures
Authors: Bregante Monica , Gambale Franco , LoSchiavo Fiorella
Published: April 2003

Ionic transport properties of protoplasts obtained from embryogenic carrot suspension cells were studied by the patch‐clamp technique. In the whole‐cell configuration, carrot protoplasts presented macroscopic time‐dependent outward currents, showing kinetics of activation which did not depend appreciably on the amplitude of the stimulus. Time‐ and voltage‐dependent whole‐cell inward rectifying currents as well as instantaneous non‐selective currents were also observed. Both time‐dependent inward and outward currents are carried by potassium ions. In a cell‐attached configuration, two types of single‐channel signals, displaying conductances of 10 and 17 pS, were observed; the instantaneous 10 pS channel was also present in outside‐out excised patches.
Ras‐interacting domain of RGL blocks Ras‐dependent signal transduction in Xenopus oocytes
Authors: Koyama Shinya , Chen Yen-Wen , Ikeda Masahiro , Muslin Anthony J. , Williams Lewis T. , Kikuchi Akira
Published: April 2003

RalGDS family members (RalGDS and RGL) interact with the GTP‐bound form of Ras through its effector loop. The C‐terminal region (amino acids 602–768) of RGL is responsible for binding to Ras. In this paper we characterized a Ras‐interacting domain of RGL using deletion mutants of RGL(602–768). RGL(602–768), RGL(632–768), and RGL(602–734) bound to the GTP‐bound form of Ras and inhibited the GAP activity of NF‐1. RGL(646–768) showed a low binding activity to Ras and inhibited GAP activity of NF‐1 weakly. None of RGL(659–768), RGL(685–768), RGL(602–709), and RGL(602–686) bound to Ras or inhibited GAP activity of NF‐1. These results indicate that amino acids 632–734 of RGL constitute a nearly minimal domain that contains the binding element for Ras. RGL(632–734) inhibited v‐Ras‐ but not progesterone‐induced Xenopus oocyte maturation. Furthermore, RGL(632–734) inhibited v‐Ras‐ but not v‐Raf‐dependent extracellular signal‐regulated kinase activation in Xenopus oocytes. These results clearly demonstrate that the Ras‐interacting domain of RGL is important for Ras‐dependent signal transduction in vivo.
Carbohydrate induced modulation of cell membrane: II. Spin label study of fluidity changes in peripheral blood lymphocyte membrane
Authors: Hajela Krishnan , Kayestha Rajeev , Sumati
Published: April 2003

This paper reports for the first time, that binding of various mono‐, di‐, and trisaccharides to membrane lectins reduces the rotational motion of membrane proteins and lipids indicating a decrease in membrane fluidity as studied by EPR spectroscopy using spin probes. Interaction of polysaccharides with lymphocyte resulted in an extensive decrease in membrane fluidity making the membrane almost rigid. The decrease in fluidity was dose‐dependent, dependent on the multivalency of the ligand used, and was sensitive to presence of EDTA and sodium azide. Binding of two different carbohydrate ligands on their respective surface lectins has a synergistic effect on the decrease in membrane fluidity.
Structure of fuscopeptins, phytotoxic metabolites of Pseudomonas fuscovaginae
Authors: Ballio A. , Bossa F. , Camoni L. , Di Giorgio D. , Flamand M.-C. , Maraite H. , Nitti G. , Pucci P. , Scaloni A.
Published: April 2003

The structure of the fuscopeptins, bioactive lipodepsipeptides produced in culture by the gramineae pathogen Pseudomonas fuscovaginae, has been determined. The combined use of FAB mass spectrometry, NMR spectroscopy and chemical and enzymatic procedures allowed one to define a peptide moiety corresponding to ZDhb‐DPro‐LLeu‐DAla‐DAla‐DAla‐DAlaDVal‐Gly‐DAla‐DVal‐DAla‐DVal‐ZDhb‐DaThr‐LAla‐LDabDDab‐LPhe with the terminal carboxyl group closing a macrocyclic ring on the hydroxyl group of the allothreonine residue. The N‐terminus is in turn acylated by 3‐hydroxyoctanoate in fuscopeptin A and 3‐hydroxydecanoate in fuscopeptin B. Some preliminary data on the biological activity of fuscopeptins are also reported.
The free radical site in pea seedling copper amine oxidase probed by resonance Raman spectroscopy and generated by photolysis of caged substrate
Authors: Johnson Steven A. , Bisby Roger H. , Tavender Susan M. , Parker Anthony W.
Published: April 2003

Resonance Raman spectra were obtained of the free radical site in substrate reduced anaerobic samples of pea seedling copper amine oxidase (PSAO). The spectra differ significantly from those reported previously for E. coli copper amine oxidase [Moenne‐Loccoz et al. (1995) Biochemistry 34, 702]. The spectra were found to be independent of substrate (benzylamine, spermidine or methylamine) used to reduce the TOPA quinone cofactor, however, several of the peaks in the Raman spectrum displayed small shifts on using [15N]benzylamine, proving incorporation of the substrate nitrogen atom onto the cofactor radical. Changes in the spectrum were also observed when measured in D2O solution indicating a strongly bound proton in the radical. The spectra were independent of pH values between 5 and 9 and are interpreted as showing that the radical exists as a semiiminoquinone radical monoanion. Benzylamine and phenethylamine have been caged with 2‐nitrobenzaldehyde and shown by laser flash photolysis to uncage on a sub‐millisecond timescale. Preliminary experiments have shown the formation of the enzyme radical intermediate on laser flash photolysis of 2‐nitrobenzyl‐caged benzylamine in the presence of enzyme. This should permit time‐resolved resonance Raman spectral investigations of the catalytic cycle of copper amine oxidases.
Identification of phosphatidate phosphohydrolase purified from rat liver membranes on SDS‐polyacrylamide gel electrophoresis
Authors: Siess E.A. , Hofstetter M.M.
Published: April 2003

Phosphatidate phosphohydrolase (PAP; EC 3.1.3.4) insensitive to N‐ethyleimide was partially purified from rat liver membranes by a combination of chromatographic methods, immunoabsorption and glycerol gradient centrifugation. The specific activity was increased more than 600‐fold over that of the membrane extract. Enzyme antibodies precipitating more than 80% of PAP were obtained and used for the identification of PAP protein on SDS‐polyacrylamide gels employing the immunodetection method of Muilerman et al. [(1982) Anal. Biochem. 120, 46–51]. By this approach PAP was localized as a 31 kDa polypeptide.
An 11.8 kDa proteolytic fragment of the E. coli trigger factor represents the domain carrying the peptidyl‐prolyl cis/trans isomerase activity
Authors: Stoller Gerlind , Tradler Thomas , Rücknagel Karl P. , Rahfeld Jens-U. , Fischer Gunter
Published: April 2003

The 48 kDa trigger factor (TF) of E. coli was shown to be a peptidyl‐prolyl cis/trans isomerase (PPIase). Its location on a ribosomal particle is unique among the PPIases described so far, and suggests a role in de novo protein folding. The trigger factor was investigated with regard to a domain carrying the catalytic activity. An enzymatically active fragment could be isolated after proteolysis by subtilisin. The resulting polypeptide was analysed by N‐terminal sequencing and MALDI‐TOF mass spectrometry revealing an 11.8 kDa fragment of TF encompassing the amino acid residues Arg‐145 to Glu‐251. The nucleotide sequence encoding the amino acid residues Met‐140 to Ala‐250 of the TF was cloned into vector pQE32. After expression in E. coli the resulting His‐tagged polypeptide was isolated on an Ni2+‐NTA column. Subsequent digestion with subtilisin and anion‐exchange chromatography yielded a TF fragment encompassing amino acids Gln‐148 to Thr‐249. This fragment may represent the catalytic core of TF since PPIase activity with a specificity constant k

cat
/K

m
of 1.3 μM−1 s−1 could be demonstrated when using Suc‐Ala‐Phe‐Pro‐Phe‐NH‐Np as a substrate. Moreover, as was observed for the complete, authentic TF the PPIase activity of the fragment was not inhibited by the peptidomacrolide FK506.
Identification of cyclic AMP‐phosphodiesterase variants from the PDE4D gene expressed in human peripheral mononuclear cells
Authors: Némoz Georges , Zhang RuoBo , Sette Claudio , Conti Marco
Published: April 2003

To determine whether the expression of different PDE4D variants is unique to the rat or conserved through evolution, we have characterized the different PDE4D mRNAs expressed in human peripheral blood mononuclear cells. RT‐PCR was performed using primers based on rat sequences and mRNAs from mononuclear cells. The specifically amplified fragments had a size identical to that predicted for rat PDE4D1, PDE4D2 and PDE4D3. Sequencing confirmed that these fragments are derived from the human PDE4D gene. Their sequence was highly homologous to that reported for the rat variants. cDNAs corresponding to the entire ORF of human PDE4D2 and PDE4D3 were expressed in mammalian cells, causing a large increase in PDE activity. Western blot analysis of human peripheral blood mononuclear cell extracts demonstrated the presence of proteins corresponding to the recombinant PDE4D1 and PDE4D2. The pattern of splicing and different promoter usage of the PDE4D gene is therefore conserved during evolution, which indicates an important physiological role.
Inhibition of astrocyte gap junctional communication by ATP depletion is reversed by calcium sequestration
Authors: Vera Begoña , Sánchez-Abarca Luis I. , Bolaños Juan P. , Medina Jose M.
Published: April 2003

We have studied the possible role of cellular energy status in the regulation of gap junction permeability in rat astrocytes in primary culture. Incubation with the mitochondrial respiratory chain inhibitor antimycin (5 ng/ml) for 16 h caused a significant decrease in ATP concentrations. This effect was accompanied by a dose‐dependent inhibition of gap junction permeability as assessed by the scrape‐loading/Lucifer yellow transfer technique. No cell death was observed following this treatment. Restoration of cellular ATP levels by a further 24 h incubation in antimycin‐free medium reversed the inhibition of Lucifer yellow transfer caused by antimycin. The inhibition of Lucifer yellow transfer brought about by antimycin treatment was also reversed by a short incubation of the cells with the calcium chelator EGTA plus the calcium ionophore A23187. These results suggest that ATP depletion causes a reversible inhibition of gap junction permeability through a calcium‐mediated mechanism.
Formation of polymeric chelate bridges between double‐stranded DNA molecules fixed in spatial structure of liquid‐crystalline dispersions
Authors: Yevdokimov Yu.M. , Salyanov V.I. , Gedig E. , Spener F.
Published: April 2003

The formation of cholesteric liquid‐crystalline dispersions from DNA‐daunomycin complexes in water‐salt polyethyleneglycol‐containing solutions was investigated. In the case of nonclassical complex formation between DNA and daunomycin (DAU), reactive groups of DAU were used for the formation of polymeric chelate complex with divalent copper ions (‐DAU‐Cu‐…‐Cu‐DAU‐), located between neighboring double‐stranded DNA molecules, fixed in spatial structure of liquid‐crystalline dispersions. The formation of polymeric chelate complex does not depend upon the sense of helicoidal twist of DNA cholesterics. A many‐fold increase in the CD band in the DAU absorption region is specific to this process. A reduction of the divalent copper ions as a result of a redox‐process is accompanied by destroying of structure of polymeric chelate complex between DNA molecules and by disappearance of the abnormal CD band in daunomycin absorption region.
A rise in nuclear calcium translocates annexins IV and V to the nuclear envelope
Authors: Raynal Patrick , Kuijpers Gemma , Rojas Eduardo , Pollard Harvey B.
Published: April 2003

Following incubation of human fibroblasts with Ca2+ ionophore A23187, we found strong immunofluorescence labelling of the nuclear envelope by annexin IV antibody. Using confocal imaging of cells loaded with Fluo‐3, we showed that A23187 generates an intense and sustained rise of Ca2+ in the nucleus. By contrast, stimulation without extracellular Ca2+ produces only a brief rise in nuclear Ca2+ that does not promote annexin IV translocation to the nuclear envelope, and compounds that induce only a transient increase of nuclear Ca2+ do not support translocation of annexin IV. In addition, annexin V was also translocated to the nuclear envelope by A23187, but distribution of annexins I, II, VI and VII is unaffected. In in vitro assays with isolated nuclei, annexin V was also found to bind to the nuclear envelope in a Ca2+‐dependent manner. These results demonstrate that the translocation to the nuclear envelope of different types of Ca2+‐regulated proteins is directly triggered by a major rise of Ca2+ in the nucleus.
Identification of nonconserved amino acids in the AT1 receptor which comprise a general binding site for biphenylimidazole antagonists
Authors: Nirula Vaneet , Zheng Wei , Krishnamurthi Kamakshi , Kathryn Sandberg
Published: April 2003

Mutational analysis based on pharmacological differences between mammalian and amphibian angiotensin II receptors (AT receptors) previously led to construction of a mutant receptor that gained >25000‐fold affinity for the biphenylimidazole, Losartan. This variant frog receptor also bound with high affinity other nonpeptides in the biphenylimidazole chemical class according to the following rank order of potency (expressed in Fmut values = mutant IC50/rAT1b IC50): Losartan, 0.91; L‐162,389, 1.0; L‐163,491, 1.9; L‐158,809, 3.5; L‐163,017, 3.9; SC‐51,316, 3.9. In contrast, the imidazoleacrylic acids, SKF‐108,566 (Fmut = 160) and SB‐203,220 (Fmut = 170), bound with markedly less affinity. Thus, nonconserved residues determining the molecular requirements for biphenylimidazole recognition are conserved in general, but are not identical to nonconserved residues necessary for high affinity binding of imidazoleacrylic acids.