bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and expression of the ε4 subunit of the NMDA receptor channel
Authors: Ikeda Kazutaka , Nagasawa Michiaki , Mori Hisashi , Araki Kazuaki , Sakimura Kenji , Watanabe Masahiko , Inoue Yoshiro , Mishina Masayoshi
Published: July 2002

The primary structure of a novel subunit of the mouse NMDA (N‐methyl‐d‐aspartate) receptor channel, designated ε4, has been revealed by cloning and sequencing the cDNA. The ε4 subunit shares high amino acid sequence identity with the ε1, ε2 and ε3 subunits of the mouse NMDA. receptor channel, thus constituting the ε subfamily of the glutamate receptor channel. Expression from cloned cDNAs of the ε4 subunit together with the ζ1 subunit in Xenopus oocytes yields Functional NMDA receptor channels. The ε4/ζ1 heteromeric channel exhibits high apparent affinities for agonists and low sensitivities to competitive antagonists. The ε4 subunit is thus distinct in Functional properties from the ε1, ε2 and ε3 subunits, and contributes further diversity of the NMDA receptor channel.
Human immunodeficiency virus type 1 productive infection in staurosporine‐blocked quiescent cells
Authors: Aranda-Anzaldo A. , Viza D.
Published: July 2002

Staurosporine, an antibiotic known to inhibit cellular protein kinases, can reversibly block the progress of normal and tumour cells into the cell cycle. The ability of HIV‐1 to infect and replicate in cells blocked by staurosporine was investigated. The results show that blocked, non‐cycling cells can be productively infected by HIV‐1, steadily releasing infectious progeny virus for several weeks. This suggests that at least in some cases, HIV‐1 can be found in a stable and active state in resting, non‐proliferating T cells.
Ring finger in the peroxisome assembly factor‐1
Authors: Patarca Roberto , Fletcher Mary Ann
Published: July 2002

The peroxisome assembly factor‐1 (PAF‐1) is reported here to contain the signature subsequence for a ring finger motif in its carboxyl‐terminus. This conserved subsequence in PAF‐1 may be the key to a gene expression regulatory pathway important in peroxisome biogenesis.
Effect of dibutyryl cyclic AMP and isoproterenol on 7β‐hydroxycholesterol cytotoxicity and esterification in spontaneous transformed cell lines derived from astrocyte primary cultures
Authors: Behr Patrick , Kupferberg Alexandre , Leray Claude , Schellenbaum Laurence , Urban Paul-Francis , Vincendon Guy , Mersel Marcel
Published: July 2002

Incubation of spontaneous transformed cells derived from astrocyte primary cultures with 30 μM 7β‐hydroxycholesterol (7β‐OH‐CH) which is lethal to the cells or with 150 μM isoproterenol reduces the intracellular level of cAMP (4‐ and 2‐fold respectively). Treatment of the cultures with 0.5 mM dibutyryl (db)‐cAMP and 7β‐OH‐CH increases 3‐fold the intracellular level of cAMP and both, db‐cAMP and isoproterenol, raise the lethal effect of 7β‐OH‐CH and its esterification on C‐3‐OH by naturally occurring fatty acids (metabolite). Kinetics studies of net steryl‐3‐esters hydrolysis revealed that db‐cAMP and isoproterenol lower that of cholesteryl‐3‐esters (2‐fold) whereas the opposite is found for the metabolite. These data demonstrate that (i) high cAMP intracellular levels modulate differently the net hydrolysis of cholesteryl‐3‐esters and metabolite, (ii) isoproterenol acts otherwise than cAMP on 7β‐OH‐CH esterification, (iii) the cytotoxicity of 7β‐OH‐CH is linked to its own esterification. The accumulation of metabolite subsequent to db‐cAMP or isoproterenol treatment as a result of acyl‐CoA:cholesterol acyl transferase activation is discussed.
Ciliary neurotrophic factor induces acute‐phase protein expression in hepatocytes
Authors: Schooltink Heidi , Stoyan Tanja , Roeb Elke , Heinrich Peter C. , Rose-John Stefan
Published: July 2002

During inflammatory states, hepatocytes are induced to synthesize and secrete a group proteins called acute‐phase proteins. It has recently been shown that besides interleukin‐6 (IL‐6) related cytokines such as leukemia inhibitory factor, oncostation M and interleukin‐11 are also mediators of the degatic acute‐phase response. All these mediators belong to the dematoqaietic family of α‐helical cytokines. Here we show that an additional member of this cytokine family, ciliary neurotrophic factor (CNTF), induces the hepatic acute‐phase protein genes heptaglobin α1‐antichymotrypsin, α2‐macroglobulin and β‐fibrinogen in human hepatoma cells (HepG2) and in primary rat hepatocytes with a time course and dose‐response comparable with that of IL‐6. Our next aim was to define the receptor components used by CNTF on hepatic cells. Using a cell‐free binding assay we exclude that CNTF binds to the 80 kDa IL‐6 receptor, a protein with significant homology to the CNTF receptor which has recently been cloned from neuroblastoma cells. In human hepatoma cells (Hep3B) which lack the leukemia inhibitory factor receptor. CNTF was not able to induce acute‐phase protein synthesis, indicating that this receptor protein may be part of the functional CNTF receptor on hepatic cells.
Kinetic mechanism of ketoreductase activity of prostaglandin F synthase from bovine lung
Authors: Barski Oleg A. , Watanabe Kikuko
Published: July 2002

The kinetic mechanism of ketoreductase activity of bovine lung prostaglandin F synthase, expressed in E. coli, was investigated. Data on initial velocity and radioisotope exchange between [3H]prostaglandin D; and 9α, 11β‐prostaglandin F2 suggest that the enzyme obeys the ping‐pong mechanism. Using a fluorescence technique we obtained a binding constant of 3 μM for NADPH. This is in close correlation with the kinetically determined intrinsic Michaelis constant for NADPH. Activation energy of the redox process was determined from the temperature dependence of maximal velocities for nitrobenzaldehyde and menadione and was found to be 119 and 96 kJ/mol, respectively.
Nuclear protein transport is functionally conserved between yeast and higher eukaryotes
Authors: Wagner Philipp , Hall Michael N.
Published: July 2002

The ability of a yeast nuclear protein to be transported into the nucleus of a higher eukaryotic cell was investigated. Mcm1, a transcriptional activator protein from Saccharomyces cerevisiae, was microinjected into the cytoplasm of Xenopus laevis frog oocytes. Mcm1 was imported into the oocyte nucleus indicating that the machinery for nuclear transport is conserved from yeast to higher eukaryotes. Furthermore, by comparing the nuclear import of free proteins and protein‐gold complexes, we found that protein‐gold complex formation appears to partially and specifically inactivate the nuclear transport activity of Mcm1 and that nucleoplasmin is an exceptionally good nuclear import substrate.
An interaction of β‐amyloid with aluminium in vitro
Authors: Exley Christopher , Price Nicholas C. , Kelly Sharon M. , Birchall J.Derek
Published: July 2002

We have used circular dichroism spectroscopy to confirm that, in a membrane‐mimicking solvent, AβP(1–40) adopts a partially helical conformation and we have demonstrated the loss of this structure in the presence of physiologically relevant concentrations of aluminium. This is the first evidence of a direct biochemical interaction between aluminium and β‐amyloid and may have important implications for the pathogenesis of Alzheimer's disease.
Glucuronidation of thyroid hormone by human bilirubin and phenol UDP‐glucuronyltransferase isoenzymes
Authors: Visser Theo J. , Kaptein Ellen , Gijzel Anthonie L. , de Herder Wouter W. , Ebner Thomas , Burchell Brian
Published: July 2002

The glucuronidation of thyroid hormone by UDP‐glucuronyltransferases (UGTs) stably transfected in Chinese hamster V79 lung fibroblasts was investigated. Human bilirubin UGT (HP3) and phenol UGT (HP4) both catalysed the glucuronidation of T4 and rT3, whereas glucuronidation of T3 was not significant. rT3 was the preferred substrate for both isoenzymes, glucuronidation rates being 1.6‐ and 6.4‐times higher than conjugation of T4 by HP3 and HP4 clones, respectively. This is the first identification of thyroid hormone as potential alternative endogenous substrate for bilirubin UGT.
Determination of distances involving exchangeable protons from NOESY spectra of two DNA dodecamers
Authors: Michalczyk Ryszard , Russu Irina M.
Published: July 2002

Two‐dimensional proton nuclear Overhauser effect (NOESY) spectra were obtained as a function of mixing time for two DNA dodecamers, 5'‐CGCGAATTCGCG‐3' and 5'‐CGCAAATTTGCG‐3', in aqueous solutions. The time evolution of cross‐peak volumes was quantitatively analyzed based on the approximate solution of the relaxation/exchange equation over a range of mixing times from 30 to 150 ms. Inter‐proton distances, involving exchangeable protons in key locations of the structures, were calculated and compared to the distances predicted by the crystal structures. These NMR‐derived distances enhance the number of constraints, and their accuracy, for determination of solution structures of the two DNA dodecamers.
Molecular characterization of Phl p II, a major timothy grass (Phleum pratense) pollen allergen
Authors: Dolecek Christiane , Vrtala Susanne , Laffer Sylvia , Steinberger Peter , Kraft Dietrich , Scheiner Otto , Valenta Rudolf
Published: July 2002

Grass pollen allergens belong to the most important and widespread elicitors of pollen allergy. Using serum IgE from a grass pollen allergic patient, a complete cDNA encoding a group II allergen was isolated from a timothy grass (Phleum pratense) pollen expression library. The deduced amino acid sequence of the Phl p II allergen shows an average sequence identity of 61% with the protein sequences determined for group II/III allergens from rye grass (Lolium perenne) and a sequence identity of 43% with the C‐terminal portion of group I grass pollen allergens from different species. A hydrophobic leader peptide similar to leader peptides found in other major grass pollen allergens heads the deduced amino acid sequence, indicating that group II/III grass pollen allergens belong to a family of secreted proteins. Serum IgE specific for Phl p II, detected the protein exclusively in pollen and not in other plant tissues. The recombinant Phl p II was expressed in Escherichia coli and showed similar IgE‐binding capacity as the natural allergen.
Identification of a new b‐type cytochrome from the whitefish Coregonidae eggs
Authors: Valyushok Dmitry S. , Bykhovsky Vladimir Ya. , Arutjunjan Alexander M. , Artzatbanov Vladislav Yu.
Published: July 2002

A new b‐type cytochrome, termed cytochrome b
560 according to the wavelength maximum of its α‐band in the reduced minus oxidized difference spectrum, was isolated from eggs of whitefish and partially purified by fractionation of the water‐soluble moiety of yolk. The absolute absorption spectrum of reduced cytochrome b
650 has maxima at 426, 529 and 560 nm and that of the oxidized form at 416 nm. The reduced minus oxidized difference spectrum has maxima at 428, 529 and 560 nm. The midpoint potential of this cytochrome is +193 mV. Based on the MCD spectra of reduced cytochrome and the optical absorption spectra in the visible region of the oxidized cytochrome, it is suggested that the heme iron in cytochrome b
560 has two histidine imidazoles as the 5th and 6th axial ligands.
Transport of prepro‐albumin into inverted vesicles prepared from the inner membrane of rat liver mitochondria
Authors: Ono Hideyu , Yoshida Tadashi , Tuboi Syozo
Published: July 2002

When inverted vesicles prepared from the inner membrane of rat liver mitochondria were incubated with prepro‐rat serum albumin, considerable amounts of prepro‐albumin and pro‐albumin were recovered with the inverted vesicles re‐isolated by centrifugation. Pro‐albumin was resistant to trypsin, but prepro‐albumin was completely digested by trypsin, indicating that prepro‐albumin was transported into the vesicles and concomitantly converted to pro‐albumin. This transport process required ATP, but not a membrane potential. These results suggest that some export machinery for a protein having an amino acid sequence in its N‐terminal portion similar to the signal sequence of secretory protein exists in the inner mitochondrial membrane.
Extensive 1H NMR resonance assignment of proteins using natural abundance gradient‐enhanced 13C−1H correlation spectroscopy
Authors: Medvedeva Svetlana , Simorre Jean-Pierre , Brutscher Bernhard , Guerlesquin Françoise , Marion Dominique
Published: July 2002

The reliability and completeness of 1H NMR resonance assignment can be improved by the use of 13C−1H HSQC correlation spectra on unlabelled protein samples using pulsed field gradients. This technique is illustrated on a 5.2 mM sample of the 79 residue Desulfovibrio vulgaris ferrocytochrome c
553. Protons attached to the same carbon can be unambiguously paired in a HSQC spectrum. Contrary to 1H, most amino acids exhibit characteristic 13C chemical shift ranges, which can be used for 13C assignment. This technique is especially useful for long side chain residues, such as Gln, Glu, Lys, Arg.
Antibodies to fluorylsulfonylbenzoyladenosine permit identification of protein kinases
Authors: Parker Peter J.
Published: July 2002

Antibodies were raised to fluorylsulfonylbenzoyladenosine (FSBA) coupled to carrier proteins. Affinity purified sera are shown to specifically detect protein kinase C‐β1 treated with FSBA. Immunodetection is specific to modification at the ATP binding site as judged by the ability of ATP to block subsequent detection. The antisera are shown to react with a number of U937 cell proteins fractionated on Mono Q and treated with FSBA; the majority of these immunoreactions are prevented by inclusion of ATP in the FSBA treatments. One of these is positively identified as PKC‐β1.
Dramatic changes in control properties that accompany channelling and metabolite sequestration
Authors: Kholodenko Boris N. , Cascante Marta , Westerhoff Hans V.
Published: July 2002

A simple summation theorem describes the control of fluxes in ‘ideal’ metabolic pathways. This paper shows how this theorem and the control properties of a pathway change when direct transfer of intermediates and/or sequestration of metabolites involved in moiety conservations (by enzymes present at high concentrations) take place. The derived generalized summation theorem quantifies the extent to which metabolite sequestration decreases and direct metabolite transfer can increase the control exerted by enzymes on the flux. The implications of metabolite channelling for the control of fluxes are discussed quantitatively.
Purification of an UDP‐glucose:flavone, 7‐O‐glucosyltransferase, from Silene latifolia using a specific interaction between the enzyme and phenyl‐sepharose
Authors: Vellekoop P. , Lugones L. , van Brederode J.
Published: July 2002

An UDP‐glucose:flavonoid, 7‐O‐glucosyltransferase, from Silene latifolia was isolated from petals and purified 450‐fold using a combination of gel‐filtration, affinity chromatography and anion‐exchange chromatography. Affinity chromatography on a phenyl‐Sepharose CL‐4B column in combination with elution with the substrate, isovitexin (6‐C‐glucosylapigenin), was an especially effective purification step. A purification factor between 10 and 20 could be reached using this column. A possible mechanism for the specific interaction of the enzyme with the phenyl‐Sepharose will be discussed. This method of purification may also be applicable to other enzymes which use aromatic compounds as substrates. On a SDS‐PAGE gel a band of 54 kDa, which co‐purified with enzyme activity, could be detected in the purest fraction.
Analysis of a mutant amino acid‐activating domain of surfactin synthetase bearing a serine‐to‐alanine substitution at the site of carboxylthioester formation
Authors: Vollenbroich Dirk , Kluge Britta , D'Souza Cletus , Zuber Peter , Vater Joachim
Published: July 2002

The reactive serine of the TGGHSL thioester binding motif of the first amino acid‐activating domain of surfactin synthetase was replaced by alanine using site‐directed mutagenesis. The multienzyme from cells of the resulting mutant lost its ability for thioester formation with l‐Glu and was therefore inactive in surfactin production. The thiolation reactions catalyzed by the other amino acid‐activating domains of surfactin synthetase were not affected by the mutation. The results show that l‐Glu is acativated at the first domain of surfactin synthetase, and give further evidence that a serine residue is essential for substrate amino acid activation at the reaction centers of peptide synthetases.
Identification and differential expression of yeast SEC23‐related gene (Msec23) in mouse tissues
Authors: Wadhwa Renu , Kaul Sunil C. , Komatsu Yasuhiko , Ikawa Yoji , Sarai Akinori , Sugimoto Yoshikazu
Published: July 2002

We have isolated a yeast SEC23‐related clone (Msec23) from mouse fibroblast cDNA library. It has an open reading frame of 1721 bp (64% homologous to SEC23‐2.3 kb) which can potentially encode a 64.7 kDa protein (61% homologous to 85.4 kDa product of SEC23). The deduced Msec23 protein (Msec23p) sequence contains three successive Ig‐like domains at the N‐terminus followed by amphipathic α‐helical regions, suggesting the potential of Msec23p to interact with other protein components. Further, Msec23 is differentially expressed in mouse tissues with its high level in brain and fibroblasts.
A novel nucleosome assembly procedure (with a little help from pectin)
Authors: Sobolewski Christopher H.M. , Klump Horst H. , Lindsey George G.
Published: July 2002

The acidic polysaccharide pectin (α‐1,4‐polygalacturonic acid) has been introduced as a nucleosome assembly facilitator as a substitute for polyglutamic acid. The pectin‐assembled nucleosomes were indistinguishable from polyglutamic acid‐assembled nucleosomes by thermal denaturation and DNAse I digestion. Pectin had two major advantages over polyglutamic acid ‐ the yield of assembled cores was approximately 50% higher and the pectin could be easily removed after completion of the reassembly procedure by dialysis following pectinase cleavage.
Regulation of cardiac insulin receptor function by guanosine nucleotides
Authors: Russ Martina , Reinauer Hans , Eckel Jürgen
Published: July 2002

The present study examined the effect of GTP‐γ‐S on the function of insulin receptors partially purified from adult rat cardiomyocytes by WGA chromatography. GTP‐γ‐S increased receptor autophosphorylation about two times and fully mimicked the stimulatory action of insulin on poly(Glu:Tyr) phosphorylation with no additional effect of the hormone. The effect of GTP‐γ‐S was specific, dose‐dependent, and due to an increase in the V
max of the kinase. In the presence of ATP or AMP‐PNP, insulin significantly enhanced the binding of [5S]GTP‐γ‐S to the partially purified insulin receptor. The findings suggest coupling of the insulin receptor to a G‐protein which may be involved in the regulation of tyrosine kinase activity.
Length polymorphisms in new human collagen‐like loci
Authors: Doering Jeffrey L. , Burket Anne E. , Vogel Lawrence C.
Published: July 2002

Two new collagen‐like loci have been identified in the human genome which have sequence similarity to the triple‐helical coding region of the pro‐α2(I) gene. Both loci exhibit length polymorphism due to alleles that contain deletions. The deletion at one locus is 400 bp while the deletion at the second locus is 200 bp. The second locus is on chromosome 17 and its two alleles are not in Hardy‐Weinberg equilibrium. These loci are candidates for involvement in connective tissue disorders.
Recoverin mediates the calcium effect upon rhodopsin phosphorylation and cGMP hydrolysis in bovine retina rod cells
Authors: Gorodovikova Elena N. , Gimelbrant Alexandr A. , Senin Ivan I. , Philippov Pavel P.
Published: July 2002

Rhodopsin phosphorylation and in consequence cGMP hydrolysis in bovine rod outer segments are Ca2+ dependent in the presence of ATP. The level of rhodopsin phosphorylation decreases and the lifetime of active phosphodiesterase increases when the free [Ca2+] is raised from <1 nM to about 1 μM; in both cases the half‐maximal effect was observed at 140–170 nM of free Ca2+. Antibodies to recoverin reverse both effects at high [Ca2+] but have no influence at low [Ca2+]. We conclude that the Ca2+ effects observed are mediated by recoverin which inhibits rhodopsin kinase at a high Ca2+ level.
The pH dependent spectral properties of Clostridium pasteurianum 2[Fe4S4] ferredoxin
Authors: Calzolai Luigi , Messori Luigi , Monnanni Roberto
Published: July 2002

The recently assigned 1H NMR hyperfine signals of Clostridium pasteurianum ferredoxin were investigated over the pH range 8–12 to monitor possible pH‐dependent conformational changes of the protein. For very high pH values minor perturbations were detected in the chemical shifts of three signals assigned to β‐CH2, cysteine protons of cluster II, while cluster I was not affected at all. These chemical shift variations, which can be fitted to a single pK
a ≈ 10.9, are interpreted as an effect of deprotonation of the phenolic group of Tyr‐2, located reasonably close to cluster II. This hypothesis has been supported by means of other techniques such as CD and absorption spectroscopy that, on turn, are able to reveal minor pH‐dependent spectral variations at high pH. Finally a UV difference experiment has provided further evidence for deprotonation of the phenolic group of Tyr‐2. The possible influence of deprotonation of Tyr‐2 on the redox properties of cluster II is discussed.
The human MDR3 P‐glycoprotein promotes translocation of phosphatidylcholine through the plasma membrane of fibroblasts from transgenic mice
Authors: Smith Alexander J. , Timmermans-Hereijgers Johanna L.P.M. , Roelofsen Ben , Wirtz Karel W.A. , van Blitterswijk Wim J. , Smit Jaap J.M. , Schinkel Alfred H. , Borst Piet
Published: July 2002

The mouse mdr2 P‐glycoprotein (P‐gp) and its human MDR3 homologue are present in high concentrations in the canalicular membrane of hepatocytes. Mice lacking this protein are unable to secrete phosphatidylcholine (PC) into bile, suggesting that this P‐gp is a PC translocator. We have tested this in fibroblasts from transgenic mice expressing the MDR3 gene under a vimentin promoter. Transgenic and control fibroblasts were incubated with [14C]choline to label PC. When the labeled cells were incubated with a PC transfer protein and acceptor liposomes, transfer of radioactive PC was enhanced in transgenic cells relative to the wild type controls. We conclude that the MDR3 P‐glycoprotein is able to promote the transfer of PC from the inner to the outer leaflet of the plasma membrane, supporting the idea that this protein functions as a PC flippase.
Mycotin: A lectin involved in the adherence of Mycobacteria to macrophages
Authors: Goswami Swapnanjan , Sarkar Saurav , Basu Joyoti , Kundu Manikuntala , Chakrabarti Parul
Published: July 2002

Pathogenic Mycobacteria colonize host macrophages. Attachement of these organisms to macrophages is the preliminary step prior to invasion of the macrophages by the bacteria. Western blot confirmed that walls of Mycobacterium avium and Mycobacterium tuberculosis contain molecules which are immunologically related to mycotin, a lectin found in Mycobacterium smegmatis. We have demonstrated that the adherence of Mycobacteria to macrophages is significantly inhibited by anti‐mycotin antibody or the mycotin‐specific sugar, mannan. These observations suggest that prevention of the interaction of mycotin‐related molecules on the surfaces of Mycobacteria with mannose‐specific receptors on macrophages, offers an important approach for blocking attachment of pathogenic Mycobacteria to macrophages, thereby preventing infection.
δ‐L‐(α‐Aminoadipoyl)‐L‐cysteinyl‐D‐valine synthetase: the order of peptide bond formation and timing of the epimerisation reaction
Authors: Shiau Chia-Yang , Baldwin Jack E. , Byford Michael F. , Sobey Wendy J. , Schofield Christopher J.
Published: July 2002

δ‐L(α‐Aminoadipoyl)‐L‐cysteinyl‐D‐valine (ACV) synthetase catalyses the formation of the common precursor tripeptide of both the penicillin and cephalosporin antibiotics from the l‐enantiomers of its constituent amino acids. Replacement of cysteine with l‐O‐methylserine in preparative‐scale incubations led to the isolation of both t.‐O‐methylserinyl‐L‐valine and l‐O‐methylserinyl‐D‐valine dipeptides. The dipeptides were characterized with the aid of authentic synthetic standards by both 1H NMR and electrospray ionization MS. A revised mechanism for ACV biosynthesis involving formation of the cysteinyl‐valine peptide bond before the epimerisation of valine and subsequent condensation with the δ‐carboxyl of L‐α‐aminoadipate is therefore proposed.
Cloning and characterization of the cDNA encoding rice elongation factor 1β
Authors: Matsumoto Shogo , Terui Yoshiaki , Xi Shixiong , Taira Hideharu , Ejiri Shin-ichiro
Published: July 2002

We have cloned and sequenced a cDNA coding for rice elongation factor 1β (EF‐1β). The clone was 1420 bp long and contained an open reading frame coding for 229 amino acids. The overall identity between rice EF‐1β and rice EF‐1β' [Matsumoto, S., Oizumi, N., Taira, H. and Ejiri, S. (1992) FEBS Lett. 311, 46‐48] is 60% at the amino acid sequence level; a higher percent identical residues (81%) were especially observed in the C‐temiinal region. Rice EF‐1β has no conserved phosphorylation site for casein kinase II and no leucine zipper motif, although these motifs are well conserved in EF‐1δ (= β in plants) subunits of animal EF‐1.
Identification of a chaperonin‐10 homologue in plant mitochondria
Authors: Burt W.J.E. , Leaver C.J.
Published: July 2002

Chaperonin‐60 and chaperonin‐10 form stable binary complexes in the presence of ATP. This phenomenon has been used as the basis for the identification of a chaperonin‐10 homologue in potato (Solanum tuberosum L.) mitochondria. ATP‐dependent binary complexes formed between chaperonin‐60 and chaperonin‐10 in potato mitochondrial extracts were isolated by affinity chromatography on a column of immobilised chaperonin‐60 antibodies. Partial amino acid sequence analysis of the chaperonin‐10 protein indicates that it is related in primary structure to rat mitochondrial chaperonin‐10.
Primary structure of bovine α2‐antiplasmin
Authors: Christensen Søren , Berglund Lars , Sottrup-Jensen Lars
Published: July 2002

The primary structure of bovine α2‐antiplasmin (α2AP) has been determined from cDNA and partial peptide sequencing. Mature bovine α2AP contains 470 residues and is 6 residues longer than human α2 AP. Alignment of the two protein sequences show that 81% of their amino acid residues are identically located. Bovine α2AP has 5 N‐linked carbohydrate groups, of which four are found in human α2AP (AsnlOS, 274,288 and 295). Asn227 is the fifth carbohydrate attachement site in bovine α2AP. The 3 Cys residues of bovine α2AP are present as an unpaired residue (Cys131) and as a pair in a disulfide bridge (Cys49‐Cys12). The assignment of the bridge in bovine α2AP is at variance with the previous assignment of the two disulfide bridges in human α2AP [Lijnen, H.R. et al. (1987) Eur. J. Biochem. 166, 565‐574].
Mutation of a putative ADP‐ribosylation motif in the Pasteurella multocida toxin does not affect mitogenic activity
Authors: Ward Philip N. , Higgins Theresa E. , Murphy Anne C. , Mullan Paul B. , Rozengurt Enrique , Lax Alistair J.
Published: July 2002

Pasteurella multocida toxin (PMT) is a potent mitogen for Swiss 3T3 fibroblasts and cytotoxic to embryonic bovine lung cells. Site‐directed mutagenesis was used to investigate the functional significance of a three amino acid motif in PMT that is present in five other bacterial protein toxins which exhibit ADP‐ribosyl transferase activity. Crude lysates of mutant clones were fully cytotoxic for embryonic bovine lung cells. Purified mutant toxin was also as effective at stimulating inositol phosphate turnover and nucleic acid synthesis as wild type toxin. We conclude that this motif has no functional significance in Pasteurella multocida toxin.
Regulation of the Src protein tyrosine kinase
Authors: Superti-Furga Giulio
Published: July 2002

Members of the Src family of protein tyrosine kinases are involved in a variety of cellular processes, including cell growth, cell differentiation and neuronal signalling. N‐terminal to the catalytic domain, Src family members contain a Src homology 2 (SH2) domain, a Src homology 3 (SH3) domain, and a unique domain, all capable of protein‐protein interactions. Negative regulation by phosphorylation of a conserved tyrosine residue at the C‐terminal tail of the molecules is characteristic of this family of enzymes. Phosphorylation of this residue causes the intramolecular interactions of the SH2 domain with the tail, and of the SH3 domain with an as yet undefined region, probably within the catalytic domain. Enzymatically active Src family kinases, on the other hand, are phosphorylated at a tyrosine in the middle of the catalytic domain and phosphorylation of this residue is a prerequisite for high activity. Regulators of these enzymes may thus act by altering the phosphorylation state of the two key tyrosine residues or by interfering with the regulatory intramolecular interactions, either by direct binding or by modification of the interfaces involved.
A novel myb‐related gene from Arabidopsis thaliana
Authors: Li Song Feng , Santini Joanne M. , Nicolaou Olga , Parish Roger W.
Published: July 2002

A novel myb‐like gene (Atmyb5) has been isolated from a genomic library of Arabidopsis thaliana. The gene contains a single intron in the region coding for the Myb domains. The Myb domains are highly homologous to other animal and plant Myb proteins. Arabidopsis plants transgenic for a chimeric Atmyb5 promoter/GUS gene expressed the enzyme in a developmentally controlled and tissue specific manner. The GUS activity was detected in developing leaf trichomes, stipules, epidermal cells on the margins of young rosette and cauline leaves, and in immature seeds. Atmyb5 mRNA appears between fertilization and the 16 cell stage of embryo development and persists beyond the heart stage.
Molecular cloning and functional expression of a novel rat heart P2X purinoceptor
Authors: Garcia-Guzman Miguel , Soto Florentina , Laube Bodo , Stühmer Walter
Published: July 2002

Here we describe a novel purinergic receptor, the P2X5 receptor, cloned from rat heart. The full‐length cDNA encodes a protein 455 amino acids long which shares an overall identity of 40–47% with other members of the P2X purinergic receptor family. P2X5 mRNA transcripts are found predominantly in rat heart but are also present in brain, spinal cord and adrenal gland. Functional expression of the recombinant receptor in HEK‐293 cells shows a current that resembles mostly the P2X2 phenotype: the ATP‐activated current reveals little agonist desensitization, is not activated by α,β‐meATP and is completely blocked by suramin and PPADS.
The influence of ATP on the binding of aromatic amino acids to the ligand response domain of the tyrosine repressor of Haemophilus influenzae
Authors: Kristl Sylvia , Zhao Shimin , Knappe Barbara , Somerville Ronald L. , Kungl Andreas J.
Published: July 2002

The binding of aromatic amino acids to the ligand response domain of the tyrosine repressor (TyrR) protein (TyrRlrd) of Haemophilus influenzae was investigated using circular dichroism and fluorescence spectroscopy. The induced secondary structural changes were unique for each aromatic amino acid and were further influenced by the presence or absence of ATP. Tyrosine was found to have the highest affinity for TyrRlrd in the absence of ATP, whereas the affinity for ATP itself increased in the presence of tyrosine. Binding of tyrosine is therefore the conformational trigger for the activation of TyrR whereas ATP is regarded as a conformational co‐activator.
Mg2+ induces intermembrane electron transport by cytochrome c desorption in mitochondria with the ruptured outer membrane
Authors: Lemeshko Victor V
Published: July 2002

The mechanism of external NADH oxidation in rat liver mitochondria is not clear yet, but it evidently includes the intermembrane electron transport (IMET). We showed that Mg2+ significantly stimulated IMET insensitive to rotenone–antimycin A–myxothiazol in mitochondria with the hypotonically damaged outer membrane, even in the absence of exogenous cytochrome c. This effect was more specific than simply ionic strength increase. Direct evidence for cytochrome c desorption from mitochondrial membranes under 4 mM Mg2+ influence was obtained to explain the stimulatory effect of Mg2+ on external NADH oxidation that was coupled to the inner membrane potential generation. Obtained data suggest that cytochrome b
5 is normally oriented towards the cytosol in the outer membrane, and can be accessible for endogenous cytochrome c reduction only through the outer membrane rupture or permeabilization, to activate external NADH oxidation.
Domain repertoires as a tool to derive protein recognition rules
Authors: Zucconi Adriana , Panni Simona , Paoluzi Serena , Castagnoli Luisa , Dente Luciana , Cesareni Gianni
Published: July 2002

Several approaches, some of which are described in this issue, have been proposed to assemble a complete protein interaction map. These are often based on high throughput methods that explore the ability of each gene product to bind any other element of the proteome of the organism. Here we propose that a large number of interactions can be inferred by revealing the rules underlying recognition specificity of a small number (a few hundreds) of families of protein recognition modules. This can be achieved through the construction and characterization of domain repertoires. A domain repertoire is assembled in a combinatorial fashion by allowing each amino acid position in the binding site of a given protein recognition domain to vary to include all the residues allowed at that position in the domain family. The repertoire is then searched by phage display techniques with any target of interest and from the primary structure of the binding site of the selected domains one derives rules that are used to infer the formation of complexes between natural proteins in the cell.
Further evidence that 3‐phosphoinositide‐dependent protein kinase‐1 (PDK1) is required for the stability and phosphorylation of protein kinase C (PKC) isoforms
Authors: Balendran Anudharan , Hare Gregor R , Kieloch Agnieska , Williams Michayla R , Alessi Dario R
Published: July 2002

The multi‐site phosphorylation of the protein kinase C (PKC) superfamily plays an important role in the regulation of these enzymes. One of the key phosphorylation sites required for the activation of all PKC isoforms lies in the T‐loop of the kinase domain. Recent in vitro and transfection experiments indicate that phosphorylation of this residue can be mediated by the 3‐phosphoinositide‐dependent protein kinase‐1 (PDK1). In this study, we demonstrate that in embryonic stem (ES) cells lacking PDK1 (PDK1−/− cells), the intracellular levels of endogenously expressed PKCα, PKCβI, PKCγ, PKCδ, PKCϵ, and PKC‐related kinase‐1 (PRK1) are vastly reduced compared to control ES cells (PDK1+/+ cells). The levels of PKCζ and PRK2 protein are only moderately reduced in the PDK1−/− ES cells. We demonstrate that in contrast to PKCζ expressed PDK1+/+ ES cells, PKCζ in ES cells lacking PDK1 is not phosphorylated at its T‐loop residue. This provides the first genetic evidence that PKCζ is a physiological substrate for PDK1. In contrast, PRK2 is still partially phosphorylated at its T‐loop in PDK1−/− cells, indicating the existence of a PDK1‐independent mechanism for the phosphorylation of PRK2 at this residue.
Analysis of trk A and p53 association
Authors: Browes Clare , Rowe Janice , Brown Anna , Montano Ximena
Published: July 2002

trk A tyrosine kinase (the high affinity receptor for nerve growth factor) binds to the p53 tumour suppressor protein in vitro and in vivo. Our aim was to determine which regions of p53 are involved in trk A association. In vitro binding experiments using baculovirus expressed trk A and in vitro transcribed and translated C‐terminus p53 deletion mutants show amino acids 327–338 critical for association. Also, analysis with mutants at the N‐terminus, conserved regions II, III, IV and V or amino acid positions 173, 175, 181, 248 and 249 (which are amino acids frequently mutated in a variety of neoplasms and transformed cell lines), show that these sites are not involved in trk A binding. Importantly, similar results are obtained after immunoprecipitation of lysates from p53 negative fibroblasts expressing trk A and the above p53 mutant proteins. These data suggest that the amino‐terminus of the oligomerisation domain of p53 is involved in p53/trk A association.
Evolutionary relationship of Rickettsiae and mitochondria
Authors: Emelyanov Victor V
Published: July 2002

Phylogenetic data support an origin of mitochondria from the α‐proteobacterial order Rickettsiales. This high‐rank taxon comprises exceptionally obligate intracellular endosymbionts of eukaryotic cells, and includes family Rickettsiaceae and a group of microorganisms termed Rickettsia‐like endosymbionts (RLEs). Most detailed phylogenetic analyses of small subunit rRNA and chaperonin 60 sequences consistently show the RLEs to have emerged before Rickettsiaceae and mitochondria sister clades. These data suggest that the origin of mitochondria and Rickettsiae has been preceded by the long‐term mutualistic relationship of an intracellular bacterium with a pro‐eukaryote, in which an invader has lost many dispensable genes, yet evolved carrier proteins to exchange respiration‐derived ATP for host metabolites as envisaged in classic endosymbiont theory.
Genistein, a soy isoflavone, is a potent α‐glucosidase inhibitor
Authors: Lee Dong-Sun , Lee Sang-Han
Published: July 2002

Genistein is an isoflavone that is known to be contained in soybean. It was proved that genistein plays a pivotal role in homeostasis in the human body. In the course of screening for useful α‐glucosidase inhibitors, we isolated and identified genistein as a candidate for α‐glucosidase inhibitor from fermentation broths of a Streptomyces sp. Genistein was shown to be a reversible, slow‐binding, non‐competitive inhibitor of yeast α‐glucosidase with a K
i value of 5.7×10−8 M when the enzyme mixture was pretreated with genistein. These results show a possibility that genistein could be a useful tool for metabolic disorders.
Rapid internalization of exogenous ganglioside GM3 and its metabolism to ceramide in human myelogenous leukemia HL‐60 cells compared with control ganglioside GM1
Authors: Nakamura Mitsuru , Tsunoda Atsuko , Furukawa Yusuke , Sakai Takao , Saito Masaki
Published: July 2002

Incorporation and metabolism of exogenous GM3 in human myelogenous leukemia HL‐60 cells were analyzed using 3H‐labeled GM3 ([3H]GM3). [3H]GM3 was rapidly internalized into the cells (trypsin‐resistant fraction) 8 times more than the control, 3H‐labeled GM1 ([3H]GM1). In addition, not only incorporation but also metabolism of [3H]GM3 was more rapid than [3H]GM1 in HL‐60 cells. Moreover, one of the metabolites was found to co‐migrate with ceramide in thin‐layer chromatography analysis and ceramide formation from exogenous GM3 is more rapid than that from exogenous GM1. These results suggested that there would be some preferential mechanism to produce ceramide from differentiation‐inducible GM3 in HL‐60 cells rather than from non‐inducing GM1.
Cloning of the fabF gene in an expression vector and in vitro characterization of recombinant fabF and fabB encoded enzymes from Escherichia coli
Authors: Edwards Patricia , Sabo Nelsen Janet , Metz James G , Dehesh Katayoon
Published: July 2002

Analysis of the β‐ketoacyl‐ACP synthase (KAS) encoded by the fabF gene of Escherichia coli has been hampered by a reported instability of the cloned gene. Here we describe biochemical characterization of purified, active protein from the recombinant fabF gene. This enzyme has the properties ascribed to KAS II and not those of a putative KAS IV reported to be encoded by fabJ, a genomic clone with DNA sequence identical to that of fabF. We also characterize active protein from a recombinant fabB gene and suggest that this method may have a general utility for analysis of KAS enzymes.
Characterization and localization of leptin receptors in the rat kidney
Authors: Serradeil-Le Gal Claudine , Raufaste Danièle , Brossard Gabrielle , Pouzet Brigitte , Marty Eléonore , Maffrand Jean-Pierre , Le Fur Gérard
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Bile salt activation of human cholesterol esterase does not require protein dimerisation
Authors: Loomes Kerry M. , Senior Hugh E.J.
Published: July 2002

Human milk cholesterol esterase (bile salt‐activated lipase) plays a role in the dietary uptake of triacylglyceride and cholesteryl ester. The activities toward these substrates are mediated through a unique bile salt‐activated mechanism. Previously, it has been proposed that a necessary step in this process is prior protein dimerisation in the presence of primary bile salts. In this study, we addressed the role of protein dimerisation by investigating bile salt interactions on full length and truncated recombinant forms, as analysed by size exclusion chromatography and concanavalin A Sepharose binding experiments. The present findings demonstrate that protein dimerisation is not an obligatory component of the bile salt‐activated pathway. A new functional role for the glycosylated C‐terminal domain in cholesterol esterase is also demonstrated in the prevention of non‐specific hydrophobic interactions.
Age‐dependent decline in the cytochrome c oxidase activity in rat heart mitochondria: role of cardiolipin
Authors: Paradies Giuseppe , Ruggiero Francesca Maria , Petrosillo Giuseppe , Quagliariello Ernesto
Published: July 2002

Cardiolipin is a major mitochondrial membrane lipid and plays a pivotal role in mitochondrial function. We have recently suggested a possible involvement of this phospholipid in the age‐linked decline of cytochrome c oxidase activity in rat heart mitochondria [G. Paradies et al. (1993) Arch. Gerontol. Geriatr. 16, 263–272]. The aim of this work was to test our earlier proposal. We have investigated whether addition of exogenous cardiolipin to mitochondria is able to reverse, in situ, the age‐linked decrease in the cytochrome oxidase activity. The method of fusion of liposomes with mitochondria developed by Hackenbrock [Hackenbrock and Chazotte (1986) Methods Enzymol. 125, 35–45] was employed in order to enrich the mitochondria cardiolipin content. We demonstrate that the lower cytochrome c oxidase activity in heart mitochondria from aged rats can be fully restored to the level of young control rats by exogenously added cardiolipin. No restoration was obtained with other phospholipids or with peroxidized cardiolipin. Our data support a key role for cardiolipin in the age‐linked decline of rat heart mitochondrial cytochrome c oxidase activity.
The S.//.A.IG amino acid motif is present in a replication dependent late H3 histone variant of P. lividus sea urchin1
Authors: Fucci Laura , Forte Amalia , Mancini Pietro , Affaitati Adele , Branno Margherita , Aniello Francesco , Geraci Giuseppe
Published: July 2002

A novel gene encoding a new H3 histone variant (H3L) has been identified in P. lividus sea urchin embryo. It encodes a H3 histone protein showing the S.//.A.IG amino acid motif typical of the replication independent H3.3 variants but in a mRNA showing the 3′ terminal stem‐loop nucleotide sequence that is typical of the replication dependent variants. The gene is intronless, the corresponding short transcript is non‐polyadenylated and its expression is replication dependent with a timing of a late variant. The new H3 variant is expressed as a minor component with respect to a major replication dependent late H3 histone here identified by partial cDNA sequence. These results show that classification of histones in replication dependent and independent variants only on the basis of their amino acid sequences should be reconsidered.
MNDA dimerizes through a complex motif involving an N‐terminal basic region
Authors: Xie Jingping , Briggs Judith A. , Briggs Robert C.
Published: July 2002

Human myeloid cell nuclear differentiation antigen (MNDA) is a myelomonocytic lineage‐specific protein that influences gene expression through interactions with other nuclear proteins and transcription factors. MNDA also self‐associates and chemical cross‐linking was used to demonstrate that MNDA forms a dimer. C‐terminal and internal deletion mutants were used to identify two regions in the N‐terminal half of MNDA essential for self‐association. One region contains an imperfect leucine zipper and the second is highly enriched in basic residues. The sequences that are essential for dimerization are separated by a highly basic amphipathic α‐helical region which was not required for dimerization.
Ozone stress modulates amine oxidase and lipoxygenase expression in lentil (Lens culinaris) seedlings1
Authors: Maccarrone Mauro , Veldink Gerrit A , Vliegenthart Johannes F.G , Finazzi Agrò Alessandro
Published: July 2002

The effect of ozone stress on polyamine metabolism and membrane lipid peroxidation in lentil seedlings through the amine oxidase and lipoxygenase activity and expression has been investigated. Ozone is shown to control the expression of these enzymes at the transcriptional level, down‐regulating the amine oxidase gene and up‐regulating the lipoxygenase gene. The decrease of amine oxidase activity correlated with the increase of putrescine concentration in the ozone‐treated plantlets, whereas the increase of lipoxygenase activity was paralleled by enhanced membrane lipid peroxidation. Finally, polyamines are shown to inhibit lipoxygenase activity in lentils.
Dual regulation of caspase activity by hydrogen peroxide: implications for apoptosis
Authors: Hampton Mark B , Orrenius Sten
Published: July 2002

The induction of apoptosis in Jurkat T‐lymphocytes with 50 μM hydrogen peroxide was associated with caspase activation. Caspase activity was first detected 3 h after treatment, and the morphological features of apoptosis were apparent by 6 h. At higher concentrations of hydrogen peroxide there was no detectable caspase activity, and the cells died by necrosis. Cells treated with hydrogen peroxide were impaired in their ability to undergo Fas‐mediated apoptosis. This appeared to be the result of direct inhibition of the cysteine‐dependent caspases. The cells were able to recover and undergo apoptosis at later times. Therefore, hydrogen peroxide has two distinct effects. It initially inhibits the caspases and delays apoptosis. Then, depending on the degree of the initial oxidative stress, the caspases are activated and the cells die by apoptosis, or they remain inactive and necrosis occurs. We discuss the physiological implications of cells having to maintain a reducing environment during apoptosis to allow the caspases to function.