bluuebunny/crossref_metadata_2025 huggingface dataset:
The human UDP‐N‐Acetylglucosamine:α‐6‐d‐Mannoside‐β‐1,2‐N‐Acetylglucosaminyltransferase II Gene (MGAT2)
Authors: Tan Jenny , D'agostaro Giacomo A. F. , Bendiak Brad , Reck Folkert , Sarkar Mohan , Squire Jeremy A. , Leong Peng , Schachter Harry
Published: July 2010

UDP‐GlcNAc:α‐6–D‐mannoside [GlcNAc to Manα1–6] β‐1,2‐N‐acetylglucosaminyltransferase II (GlcNAc‐T II, EC 2.4.1.143) is a Golgi enzyme catalyzing an essential step in the conversion of oligo‐mannbse to complex N‐glycans. A 1.2‐kb probe from a rat liver cDNA encoding GlcNAc‐T II was used to screen a human genomic DNA library in λEMBL3. Southern analysis of restriction endonuclease digests of positive phage clones identified two hybridizing fragments (3.0 and 3.5 kb) which were sub‐cloned into pBlueScript. The inserts of the resulting plasmids (pHG30 and pHG36) are over‐lapping clones containing 5.5 kb of genomic DNA. The pHG30 insert (3.0 kb) contains a 1341‐bp open reading frame encoding a 447‐amino‐acid protein, 250 bp of G+C‐rich 5′‐upstream sequence and 1.4kb of 3′‐downstream sequence. The pHG36 insert (3.5 kb) contains 2.75 kb of 5′‐upstream sequence and 750 bp of the 5′‐end of the open reading frame. The protein sequence showed the domain structure typical of all previously cloned glycosyltransferases, i.e. a short 9‐residue putative cytoplasmic N‐terminal domain, a 20‐residue hydrophobic non‐cleavable putative signal‐anchor domain and a 418‐residue C‐terminal catalytic domain. Northern analysis of human tissues showed a major message at 3 kb and minor signals at 2 and 4.5 kb. There is no sequence similarity to any previously cloned glycosyltransferases including human UDP‐GlcNAc:α‐3‐d‐mannoside [GlcNAc to Manα;1–3] β‐1,2‐N‐acetylglucosaminyltransferase I (GlcNAc‐T I) which has 445 amino acids with a 418‐residue C‐terminal catalytic domain. The human GlcNAc‐T I and II genes (MGAT1andMGAT2) map to chromosome bands 5q35 and 14q21, respectively, by fluorescencein situhybridization. The entire coding regions of human GlcNAc‐T I and II are each on a single exon. There is 92% identity between the amino acid sequences of the catalytic domains of human and rat GlcNAc‐T II. Southern analysis of restriction enzyme digests of human genomic DNA indicates that there is only a single copy of theMGAT2gene. The full‐length coding region of GlcNAc‐T II has been expressed in the baculovirus/Sf9 insect cell system, the recombinant enzyme has been purified to near homogeneity with a specific activity of about 20 μmol · min‐1· mg‐1· and the product synthesized by the recombinant enzyme has been identified by high‐resolution1H‐NMR spectroscopy and mass spectrometry.
Use of Monoclonal Antibodies in the Functional Characterization of the Saccharomyces Cerevisiae Sepl Protein
Authors: Holler Anita , Bashkirov Vladimir I. , Solinger Jachen A. , Reinhart Ursula , Heyer Wolf‐Dietrich
Published: July 2010

The Saccharomyces cerevisiae strand‐exchange protein 1 (Sepl also known as Xrnl, Keml, Rar5, Stpβ/DST2) has been demonstrated to mediate the formation of hybrid DNA from model substrates of linear double‐stranded and circular single‐stranded DNA in vitro. To delineate the mechanism by which Sep1 acts in the strand‐exchange reaction, we analyzed mouse anti‐Sep1 monoclonal antibodies for inhibition of the Sep1 in vitro activity. Of 12 class‐G immunoglobulins tested, four were found to consistently inhibit the Sep1‐mediated strand‐exchange reaction. The inhibiting antibodies were tested for inhibition of a variety of Sep1‐catalyzed DNA reactions including exonuclease activity on double‐stranded and single‐stranded DNA, renaturation of complementary single‐stranded DNA and condensation of DNA into large aggregates. All four inhibiting antibodies had no effect on the exonuclease activity of Sepl. Three antibodies specifically blocked DNA aggregation. In addition, one antibody inhibited renaturation of complementary single‐stranded DNA. This inhibition pattern underlines the importance of condensation of DNA into large aggregates in conjunction with double‐stranded DNA exonuclease activity for the in vitro homologous pairing activity of Sep1. The implications of these data for the interpretation of proteins which promote homologous pairing of DNA are discussed, in particular in light of the reannealing activity of the p53 human tumor‐suppressor protein.
Effects of the Anticonvulsant, Valproate, on the Expression of Components of the Cytochrome‐P‐450‐Mediated Monooxygenase System and GlutathioneS‐Transferases
Authors: Rogiers Vera , Akrawi May , Vercruysse Antoine , Phillips Ian R. , Shephard Elizabeth A.
Published: July 2010

It has been shown previously that the anticonvulsant agent, sodium valproate, induces certain cytochromeP‐450 monooxygenase activities and decreases glutathioneS‐transferase activity. We have used Western blotting, RNase protection assays and Northern blot hybridization to determine the effects of valproate on the abundance of individual components of the cytochromeP‐450 monooxygenase and of glutathione S‐transferase subunits. Due to the short half‐life of the drug in rats we have used anin vitroexperimental system comprised of rat hepatocytes co‐cultured with rat primitive biliary epithelial cells. Valproate was shown to be a potent inducer of two members of the cytochromeP‐450 (CYP)2B subfamily, CYP2B1 and 2B2. The induction of the proteins was mediated at the level of the mRNAs, with the mRNA for CYP2B1 being more highly induced than that for CYP2B2. The drug also induced, but to a much lesser extent, two important components of the cytochrome‐P‐450‐mediated monooxygenase system, NADPH‐dependent cytochromeP‐450 reductase and cytochromeb5, and their corresponding mRNAs. Thus, the effects of valproate on cytochromesP‐450 and other components of the cytochrome‐P‐450‐mediated monooxygenase system mimic those of another, structurally diverse, antiepileptic drug, phenobarbital. However, in contrast to phenobarbital, which induces glutathione S‐transferase subunits 1, 2, 3, 4 and 7, valproate selectively decreases the abundance of subunits 3 and/or 4. It has been shown previously that CYP2B1 is involved in the production of metabolites of valproate implicated in hepatotoxicity. The induction of this protein by valproate would thus contribute substantially to the hepatotoxic effects associated with the drug.
The Basic Subdomain of the c‐Jun Oncoprotein
Authors: Krebs Daniel , Dahmani Benamar , Antri Saïd El , Monnot Monique , Convert Odile , Mauffret Olivier , Troalen Frédéric , Fermandjian Serge
Published: July 2010

The structural properties of the basic subdomain of the basic zipper (bZIP) protein c‐Jun were examined by joint means of 1H‐NMR, CD and Fourier‐transform infrared (FTIR) spectroscopies. The basic subdomain (residues 252–281 in c‐Jun) is responsible for sequence‐specific recognition of DNA. A modified basic subdomain bSD (residues 1–35) and its N‐terminal part and C‐terminal part fragments (NP, residues 1–19; and, CP, residues 16–35) were prepared by solid‐phase synthesis and purified by HPLC. In aqueous solution, in the absence of DNA, bSD behaved mostly as an unstructured peptide characterized by only 5% a helix. However, upon mixing bSD and a specific DNA fragment, i.e. a CRE(cAMP‐responsive element)‐containing hexadecanucleotide, the α helix was stabilized to an extent of 20% at 20 °C or 35% at 2%C. At the same time, no significant change could be detected in the DNA spectra. Addition of trifluoroethanol to an aqueous bSD sample resulted in an increase of the α‐helix content so that about 60% of α helix was found at a ratio of 75% trifluoroethanol (20°C). These effects were reflected in both CD and FTIR measurements. Changes shown by the CD spectra during the process suggested a mechanism dominated by a two‐state helix/unordered transition. NMR data, namely αH chemical shifts, NOE cross‐peaks and NH temperature coefficients provided indications for extended or nascent helix structures within four short stretches dispersed along the sequence for c‐Jun bSD, contrasting with the unique and continuous stretch reported for Gcn4 (yeast general control protein 4) bSD in aqueous solution. Trifluoroethanol stabilized the α‐helix structure mainly at these four sites. The malleability of the basic subdomain of c‐Jun was emphasized in relation to its ability to fit the DNA helix in adopting an α‐helix structure. The complex formation apparently requires substantial conformational change from the peptide and only little from the oligonucleotide.
The Folding and Assembly Pathway of Tumour Necrosis Factor TNFα, a Globular Trimeric Protein
Authors: Hlodan Roman , Pain Roger H.
Published: July 2010

The mechanisms of folding and assembly of the globular, trimeric protein tumour necrosis factor‐α (TNF) were studied by chemical cross‐linking. This revealed the rapid accumulation of a dimeric intermediate. Under the conditions of renaturation used, formation of the trimer is complete within six minutes. The kinetics of change of intrinsic and 8‐anilino‐1‐naphthalene sulfonic acid fluorescence are first order and, combined with the kinetics of association, reveal the presence of folding steps both before and subsequent to formation of the trimer. Results from gel exclusion chromatography and kinetics, together with the existence of an acid‐induced molten globule, support the conclusion that TNF folds and assembles through a trimeric molten globule.
Characterization of a 2[4Fe‐4S] Ferredoxin Obtained by Chemical Insertion of the Fe‐S Clusters into the Apoferredoxin II from Rhodobacter Capsulatus
Authors: Armengaud Jean , Gaillard Jacques , Forest Eric , Jouanneau Yves
Published: July 2010

The Rhodobacter capsulatus ferredoxin II (FdII) belongs to a family of 7Fe ferredoxins containing one [3Fe‐4S] cluster and one [4Fe‐4S] cluster. This protein, encoded by the fdxA gene, has been overproduced in Escherichia coli as a soluble apoferredoxin. The purified recombinant protein was subjected to reconstitution experiments by chemical incorporation of the Fe‐S clusters under anaerobic conditions. A brown protein was obtained, the formation of which was dependent upon the complete unfolding of the polypeptide prior to incorporation of iron and sulfur atoms. The yield of the reconstituted product was higher when the reaction was carried out at slightly basic pH. The reconstituted ferredoxin was purified and shown to be distinct from the native [7Fe‐8S] ferredoxin, based on several biochemical and spectroscopic criteria. In the oxidized state, EPR revealed the quasi‐absence of [3Fe‐4S] cluster. 1H‐NMR spectroscopic analyses provided evidence that the protein was reconstituted as a 2[4Fe‐4S] ferredoxin. This conclusion was further supported by the determination by electrospray mass spectrometry of the molecular mass of the reconstituted protein, which matched within 2 Da to the mass of the FdII polypeptide incremented of eight atoms each of iron and sulfur. Exposure of the reconstituted protein to air resulted in a fast and irreversible oxidative denaturation of the Fe‐S clusters, without formation of [7Fe‐8S] form. Unlike the natural 7Fe ferredoxin, the reconstituted ferredoxin appeared incompetent in an electron‐transfer assay coupled to nitrogenase activity. The fact that the apoFdII was reconstituted as a highly unstable 8Fe ferredoxin instead of the 7Fe naturally occurring FdII is discussed in relation to the results obtained with other types of ferredoxins.
Structure of Four Acidic Oligosaccharides from the Jelly Coat Surrounding the Eggs of Xenopus Laevis
Authors: Plancke Yves , Wieruszeski Jean‐Michel , Alonso Catherine , Boilly Benoni , Strecker Gérard
Published: July 2010

Novel acidic oligosaccharides were released by reductive β‐elimination from the jelly coat eggs of the Anuran Xenopus laevis. According to the structural analysis of these oligosaccharide‐alditols, the following structures are proposed:

image

Where Kdn, 3‐deoxy‐d‐glycero‐d‐galactononulosonic acid.

These results confirm the species specificity of the glycanic structures present in the secretion of amphibian oviducts, and may form the basis of a specific egg‐sperm recognition process.
Mannosylated Lipoarabinomannan Interacts with Phagocytes
Authors: Venisse Anne , Fournié Jean‐Jacques , Puzo Germain
Published: July 2010

Infection by Mycobacterium tuberculosis first involves its adhesion to mononuclear host phagocytes. Various macrophage opsonic and non‐opsonic receptors are known to mediate this adhesion, with some specificity of mannosyl receptors for the more virulent strains. Mannosylated lipoarabinomannan, a major component of cell walls from M. tuberculosis and Mycobacterium bovis BCG, is endowed with mannooli‐gosaccharide units that could mediate its binding to these latter receptors. To explore its interaction with murine immune cells by flow cytometry, we report a new procedure to fluorescently tag the polysaccharide molecules. We covalently labeled mannosylated lipoarabinomannan from M. bovis BCG with biotin, allowing formation of stable complexes with streptavidin coupled to a fluorochrome. In this work, we demonstrated that this major carbohydrate antigen interacts selectively with murine phagocytes, i.e. granulocytes and macrophages. This binding was affected by temperature and was serum‐ and divalent‐cation‐ dependent. It also appears to involve a metabolically recycling protein receptor on the phagocyte surface and mannosyl aggretopes on the mannosylated lipoarabinomannan molecule. Thus, the latter may provide a means for mycobacteria to bind to and invade their host phagocytes. This molecule could constitute one of the early factors of mycobacterial virulence.
Photoinactivation of Photosystem II Induces Changes in the Photochemical Reaction Center II Abolishing the Regulatory Role of the Qb Site in the Dl Protein Degradation
Authors: Zer Hagit , Ohad Itzhak
Published: July 2010

The effect of 3‐(3,4‐dichlorophenyl)‐1,1 ‐dimethyl urea (diuron) binding at the secondary quinone (QB) binding site of reaction center II (RCII), on the high‐light‐induced degradation of the RCII proteins D1 and D2, and the core proteins CP43 and CP47 was investigated in vivo in Chlamydomonas reinhardtii. The degradation of the RCII‐D2 and the CP43 proteins shows a short lag relative to that of the RCII‐D1 protein. Diuron retards but does not prevent the degradation of RCII‐D1, D2 and CP43 proteins. The degradation of the CP47 protein is not retarded by diuron. The RCII‐D1 protein present in cells photoinactivated in the presence of diuron is subsequently degraded in cells transferred to low light or to darkness. The protein can be replaced (turnover) at least partially under both conditions. The RCII‐D1 protein is not degraded during photoinactivation of a cytochrome‐bf‐defective mutant. Degradation occurs however when the cells are returned to low light permitting slow reoxidation of plastoquinol [Zer, H., Prasil, O. & Ohad, I. (1994) J. Biol. Chem. 269, 17670–17676]. Addition of diuron does not prevent the degradation of the protein at this stage. Tryptic digestion of the RCII‐D1 protein is partially inhibited by diuron in isolated thylakoids [Trebst, A., Depka, B., Kraft, B. & Johanningmeier, U. (1988) Photosynth. Res. 18, 163–177] but not in thylakoids obtained from photoinactivated cells. We conclude that photoinactivation induces a series of sequential changes in RCII exposing the cleavage site of the RCII‐D1 protein to degradation and abolishing the regulatory role of the QB site occupancy by plastoquinone or analog ligands on the cleavage process. The degradation of the RCII‐D2 and CP43 proteins may be a secondary process following modification and/or loss of the RCII‐D1 protein.
Regulatory Proteolysis of the Major Light‐Harvesting Chlorophyll a/b Protein of Photosystem II by a Light‐Induced Membrane‐Associated Enzymic System
Authors: Lindahl Marika , Yang Dan‐Hui , Andersson Bertil
Published: July 2010

An endogenous proteolytic activity associated with spinach chloroplast thylakoid membranes has been identified. This enzymic activity is involved in the degradation of the major light‐harvesting chlorophyll a/b protein of photosystem II (LHCII) in response to exposure of leaves to increased irradiance. This proteolysis of LHCII requires an induction period and can only be detected 48–72 hours after transfer of the plants from low‐intensity to high‐intensity light. Once initiated by high‐intensity light, the degradation of LHCII can readily occur in complete darkness. The proteolysis can, after induction in vivo, be experimentally followed in vitro, both in isolated intact chloroplasts and thylakoid membranes. The proteolytic process is strictly dependent on ATP and the protease involved is of the serine or cysteine type. The activity can be released from isolated thylakoid membranes by washing with high concentrations of NaCl and reconstituted by readdition of the desalted wash supernatant. It is concluded that the protease is extrinsically bound to the outer surface of the stroma‐exposed regions of the stacked thylakoid membrane. The mechanism for the induction of the proteolytic process as well as its relation to previously described thylakoid proteases will be discussed.
Does Mitogen‐Activated‐Protein Kinase have a Role in Insulin Action?
Authors: Denton Richard M. , Tavaré Jeremy M.
Published: July 2010

The discovery of the mitogen‐activated protein (MAP) kinase family of protein kinases has sparked off an intensive effort to elucidate their role in the regulation of many cellular processes. These protein kinases were originally identified based on their rapid activation by insulin. In this review we concentrate on examining the evidence for and against a role for the MAP kinases Erk‐1 and Erk‐2 in mediating the effects of insulin. While there is good evidence in favour of a direct role for MAP kinase in the growth‐promoting effects of insulin and the regulation of Glut‐1 and c‐fos expression, and AP‐1 transcriptional complex activity, this is by no means conclusive. MAP kinase may also play a role in the control of mRNA translation by insulin. On the other hand, the evidence suggests that MAP kinase is not sufficient for the acute regulation of glucose transport (Glut‐4 translocation), glycogen synthesis, acetyl‐CoA carboxylase or pyruvate dehydrogenase activity. The findings suggest that insulin may utilise at least three distinct signalling pathways which do not involve MAP kinase.
Methanethiolation of the Liberated Cysteine Residues of Human α2‐Macroglobulin Treated with Methylamine Generates a Derivative with Similar Functional Characteristics as Native α2‐Macroglobulin
Authors: Jensen Poul Erik Hyldgaard , Shanbhag Vithaldas P. , Stigbrand Torgny
Published: July 2010

The thiol‐modifying reagent methyl methanethiosulfonate reacts with the cysteine residues of thiol esters released upon treatment of human α2‐macroglobulin with methylamine. This methanethiolation generates a derivative of α2‐macroglobulin, with an ‘open trap’ and slow mobility in non‐denaturing PAGE, similar to native α2‐macroglobulin. This similarity is further substantiated by surface hydrophobicity determinations and by the fact that neither the derivative nor native α2‐macroglobulin are cleared from the circulation in mice. Cleavages of bait regions in the derivative and native α2‐macroglobulin, however, result in electrophoretically fast forms which are cleared from the circulation in mice.

In contrast to native α2‐macroglobulin, which can bind 2 mol chymotrypsin/mol, α2‐macroglobulin treated with methylamine and methylmethanethiosulfonate binds only 0.8 mol chymotrypsin/mol. Protection of trypsin against inhibition by soybean trypsin inhibitor is significantly better when α2‐macroglobulin is modified by methylamine and methylmethanethiosulfonate than when it is modified by dinitrophenyl thiocyanate, which cyanylates the exposed thiol group.

The methanethiolated derivative is also more stable than the corresponding cyanylated derivative in that it is transformed to an electrophoretically fast form with a half‐life of 9 h as compared to a half‐life of 7 h for the latter. The transformation to the fast form is not due to instability of the thiol modification.
Functions of [His321]Gelsolin Isolated from a Flat Revertant of ras ‐Transformed Cells
Authors: Fujita Hisakazu , Laham Lorraine E. , Janmey Paul A. , Kwiatkowski David J. , Stossel Thomas P. , Banno Yoshiko , Nozawa Yoshinori , Müllauer Leonhard , Ishizaki Akira , Kuzumaki Noboru
Published: July 2010

A mutant gelsolin, [His321]gelsolin, was isolated from R1, a flat revertant of human activated c‐Ha‐ras oncogene‐transformed NIH/3T3 cells (EJ‐NIH/3T3) produced by ethylmethanesulfonate treatment. [His321]Gelsolin has a histidine instead of a proline residue at position 321 and suppresses the tumorigenicity of EJ‐NIH/3T3 cells when it is constitutively expressed [Müllauer, L., Fujita, H., Ishizaki, A. & Kuzumaki, N. (1993) Oncogene 8, 2531–2536]. To investigate the biochemical consequences of the amino acid substitution of His321, we expressed the [His321]gelsolin and wild‐type gelsolin in Escherichia coli, purified them, and analyzed their effects on actin, polyphosphoinositol lipids and phospholipase C. [His321]Gelsolin has decreased actin‐filament‐severing activity and increased nucleating activity compared with wild‐type gelsolin in vitro. Furthermore, compared to wild‐type gelsolin both nucleation and severing by [His321]gelsolin are inhibited more strongly by the phosphoinositol lipids phosphatidylinositol 4‐phosphate (PtdInsP) and phosphatidylinositol 4,5‐bisphosphate (PtdInsP2). In addition, [His321]gelsolin inhibits PtdInsP2 hydrolysis by phospholipase Cγ1 more strongly than wild‐type gelsolin in vitro because of its higher binding capacity for phosphoinositol lipid. Gelsolin has six homologous amino acid repeats called S1–S6. Our results suggest that the segment S3 which contains the mutation is functionally relevant for regulation of gelsolin's activities even though the relevant actin‐binding domains are in segments 1, 2, and 4–6, and that the region around the residue 321 may contain a phosphoinositol‐lipid‐binding site. Altered functions of [His321]gelsolin might be important for the loss of tumorigenicity of the ras ‐transformed cells.
Promoter Region of the Transcriptional Unit for Human α1‐Chimaerin, a Neuron‐Specific GTPase‐Activating Protein for p21rac
Authors: Dong Jing‐Ming , Smith Paul , Hall Christine , Lim Louis
Published: July 2010

α1‐chimaerin is a neuron‐specific GTPase‐activating protein for p21rac, a protein involved in morphological events. The mRNA is highly expressed in certain brain regions. It is also detected in cultured neuronal, but not in non‐neuronal cells. As a first step towards understanding the mechanisms underlying this regulation, genomic clones containing the 5′‐flanking region of the human α1‐chimaerin transcriptional unit were isolated and characterised. A cluster of multiple transcription start sites of α1‐chimaerin mRNAs was detected by primer‐extension and S1‐mapping analyses. The cluster was mapped to nucleotides −464 to −434 (relative to nucleotide A in the initiation codon) in genomic DNA. The 5′‐proximal region contained no TATA box, initiator motif and Sp1‐binding site. A 210‐bp fragment with approximately 110 bp 5′‐flanking sequence could function as a minimal promoter upon analysis using hybrid chloramphenicol acetyltransferase reporter constructs and transient transfection. Internal deletion and point‐mutation experiments revealed that a GGCCAATC sequence located at nucleotides –519 to –512 was essential for α1‐chimaerin promoter activity. Mobility‐shift assay showed the specific binding of nuclear factor(s) to this region, which was competed by the oligonucleotides corresponding to wild‐type but niot mutant forms. The data also suggest the existence of possible novel CCAAT‐binding factor(s) interacting with the α1‐chimaerin CCAAT box binding site. A cell‐type‐preferred suppressor located in the 5′‐distal region was found which may play a role in controlling neuron‐specific expression of α1‐chimaerin mRNA. These findings of a specific promoter for α1‐chimaerin transcription will facilitate further studies on its neuronal‐specific expression and function.
Induction of the Blood/Brain‐Barrier‐Associated Enzyme Alkaline Phosphatase in Endothelial Cells from Cerebral Capillaries is Mediated Via cAMP
Authors: Beuckmann Carsten , Hellwig Sabine , Galla Hans‐Joachim
Published: July 2010

The mammalian blood/brain barrier is located at the endothelial cells of the cerebral capillaries. Alkaline phosphatase is associated to a very large extent with these cells and has been established as a marker enzyme for a differentiated blood/brain barrier phenotypein vivoandin vitro.Nevertheless cultured brain capillary endothelial cells (BCEC) lose this marker enzyme because of a cessation ofde novosynthesis. Since astrocytes have been shown to possess the capability to re‐induce the enzymic activity of alkaline phosphatase in BCECin vitrowe were interested in the second messengers involved in the signal‐transduction mechanism of this induction in BCEC. For this reason we treated cultured porcine BCEC with a water‐soluble and membrane‐permeable analogue of cAMP, 8‐(4‐chlorophenylthio)‐cAMP (ClPhS‐cAMP) in the absence of astrocytes. By means of enzymic activity assays we were able to show that within three days the activity of alkaline phosphatase increased up to sixfold compared with the controls. The total activity of alkaline phosphatase in ClPhS‐cAMP‐treated BCEC was comparable to that of freshly isolated cells. Addition of cycloheximide inhibited the alkaline phosphatase activity increase. We conclude that cAMP is one of the second messengers involved in the induction of alkaline phosphatase activity in BCECin vitro.
RNase Mitochondrial RNA Processing Cleaves RNA from the Rat Mitochondrial Displacement Loop at the Origin of Heavy‐Strand DNA Replication
Authors: Tullo Apollonia , Rossmanith Walter , Imre Esther‐Maria , Sbisà Elisabetta , Saccone Cecilia , Karwan R. Michael
Published: July 2010

Ribonuclease mitochondrial RNA processing cleaves RNAs from the mammalian mitochondrial main non‐coding regulatory region, called the displacement loop. Our data demonstrate that rat cells contain a site‐specific ribonuclease mitochondrial RNA processing activity. We found that this enzyme processes the rat mitochondrial displacement‐loop RNA substrate at the level of the conserved sequence block 1, a result which is different from that for mouse. This finding correlates with the in‐vivo transcriptional analysis of the rat displacement‐loop region. Processing by homologous and heterologous ribonuclease mitochondrial RNA enzymes occurs in the same manner, suggesting a conserved mode of substrate recognition.
1H Nuclear‐Magnetic‐Resonance Investigation of Oxidized Fe4S4 Ferredoxin from Thermotoga maritima
Authors: Wildegger Gudrun , Bentrop Detlef , Ejchart Andrzej , Alber Markus , Hage Andrea , Sterner Reinhard , Rösch Paul
Published: July 2010

The oxidized Fe4S4 ferredoxin from the hyperthermophilic bacterium Thermotoga maritima has been investigated by one‐ and two‐dimensional NMR in order to characterize its hyperfine‐shifted resonances originating from the cysteinyl cluster ligands and to assign its resonances in the diamagnetic shift range. The chemical shift and relaxation time pattern of the hyperfine‐shifted signals is very similar to other oxidized Fe4S4 ferredoxins. A tentative sequence‐specific assignment of these resonances according to a general pattern of chemical shift of cysteine protons versus sequence position of cluster ligand is presented. Furthermore, sequence‐specific assignments for 85% of the amino acid residues that were obtained without any guidance by known X‐ray structures of ferredoxins are given. They reveal the formation of at least two elements of secondary structure by the polypeptide chain of T. maritima ferredoxin: an α‐helix comprising residues C43–D49 and a double–stranded antiparallel β‐sheet consisting of the N‐and C‐terminal parts of the protein. This folding pattern is very similar to that of the crystallographically characterized ferredoxin from the mesophile Desulfovibrio gigas [Kissinger, C. R., Sieker, L. C., Adman E. T. & Jensen, L. H. (1991) J. Mol. Biol. 219, 693–715] and therefore suggesting different mechanisms of stabilization for T. maritima ferredoxin and the ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus that was recently investigated by NMR [Teng, Q., Zhou, Z. H., Smith, E. T., Busse, S. C., Howard, J. B., Adams M. W. W. & La Mar, G. N. (1994) Biochemistry 33, 6316–6326].
Localization of N‐Glycosylation Sites and Functional Role of the Carbohydrate Units of GLAST‐1, a Cloned Rat Brainl‐glutamate/l‐aspartate Transporter
Authors: Conradt Marcus , Storck Thorsten , Stoffel Wilhelm
Published: July 2010

Thel‐glutamate transporter GLAST‐1 belongs to the newly discovered family of Na+‐dependent, high‐affinity glutamate transporters, which are involved in the regulation of synaptic excitatory neuro‐transmitter concentration in mammalian brain. The members of this family have a similar topological organisation with at least six transmembrane helices (TMHs) and two putative N‐glycosylation sites located in the extracellular loop connecting TMH 3 and TMH 4. Besides these two conserved N‐glycosylation motifs at Am206 and Asn216, GLAST‐1 possesses an additional one at Asn35. The putative N‐glycosylation consensus motifs (Asn‐Xaa‐Ser/Thr) were deleted by replacement of Asn206 and/or Asn216 by Thr using site‐directed mutagenesis (mutants N206T, N216T and N206,216T). The cDNAs encoding wild‐type GLAST‐1 and the three glycosylation‐defective transport proteins were expressed in theXenopus laevisoocyte system. Immunoprecipitation of the [35S]methionine‐labeled and g1ycopeptidase‐F‐treated transporter molecules indicates that GLAST‐1 is glycosylated at Asn206 and Asn216, whereas Asn35 remains unglycosylated. To assess a possible functional role of the two glycosylation sites wild‐type and glycosylation‐deficient GLAST‐1 were expressed inXenopusoocytes and characterized functionally by using the whole‐cell voltage‐clamp technique. The results prove that N‐glycosylation has no impact on the transport activity of GLAST‐1.
Characterization of the Equilibrium Between Blocked and Closed States of Muscle Thin Filaments
Authors: Head Jared G. , Ritchie Marcia D. , Geeves Michael A.
Published: July 2010

We recently proposed a three‐state model for the regulation of actomyosin interaction by tropomyosin and troponin (Tmmiddot;Tn). In this model, the thin filament exists in rapid equilibrium between the following three states: blocked, which cannot bind myosin significantly; closed, which can bind myosin weakly to form the A‐state; open, which can bind both to form the A‐state and isomerize to the strongly bound Rstate. In this study, we demonstrate that the equilibrium between the blocked and closed states is calcium sensitive with an equilibrium constant, KB, of 0.3 and ≥10 at pCa 8.9 and 4.6, respectively. The pCa dependence of KB is typical of that for calcium binding to thin filaments with a mid‐point at pCa 5.6 and a Hill coefficient of 1.8. KB is independent of ionic strength over the range 0.4–0.06 M but increases dramatically below 0.05 M to ≥10 at 0.01 M suggesting loss of the blocked state at low ionic strength. The blocked state also has reduced occupancy at high temperatures. KB, in the absence of calcium, increases from 0.2 at 5°C to 0.6 at 40°C.
Catalytic and Inhibitor‐Binding Properties of Some Active‐Site Mutants of Human Carbonic Anhydrase I
Authors: Engstrand Carina , Jonsson Bengt‐Harald , Lindskog Sven
Published: July 2010

Three isozyme‐specific residues in the active site of human carbonic anhydrase I, Va162, His67, and His200, have been changed by site‐directed mutagenesis to their counterparts in human carbonic anhydrase II, Asn62, Asn67, and Thr200. A double mutant, containing Asn62 and Asn67, and a triple mutant, containing all three alterations, were also produced. The rates of CO2 hydration and ester hydrolysis catalyzed by these mutants, the inhibition of these enzymes by the anions, SCN−, and I−, and the binding of the sulfonamide inhibitors, dansylamide and MK‐417 (a thienothiopyran‐2‐sulfonamide) have been measured. The results suggest that the effect of His200 in isozyme I is to prolong the lifetime of the enzyme‐bicarbonate complex and to increase the pKa, of the catalytic group, a zinc‐coordinated water molecule. For isozyme I, Val62 and His67 might interfere with the function of a proton ‘shuttle’ group in the active site, thus maintaining the buffer specificity of a compulsory proton‐transfer step. The single mutations have small effects on anion binding. Only the triple mutant has anion‐binding properties resembling those of isozyme II. All mutants show altered sulfonamide‐binding properties. In particular, the binding specificity is affected. While wild‐type isozyme I binds dansylamide 50 times more strongly than MK‐417, the triple mutant shows a reversed selectivity and binds MK‐417 nearly 50 times more strongly than dansylamide.
Insert Regions in Domain X of the Casein Kinase II Catalytic Subunit
Authors: Tiganis Tony , House Colin M. , Teh Trazel , Kemp Bruce E.
Published: July 2010

Casein kinase II, cyclin‐dependent kinases, and glycogen synthase kinase‐3 are members of the protein kinase subfamily with a prominent insert in domain X of their catalytic subunit sequence. The function of the insert sequence in casein kinase II was investigated utilising synthetic peptides corresponding to the insert, cross‐linking experiments, and the generation of casein kinase II insert region mutants. The mutation of basic residues (R276→A, R278→A, R281→A, K277→A) within the major insert sequence (PRFHDILQRHSRKRWERFVHSDNQHL, positions 265–290) did not affect α/β subunit association, enzyme tetramerisation, thermal stability, and peptide (RRRDDDSDDD‐NH2) phosphorylation. Similarly, replacement of residues 276–290 within the major insert with the corresponding residues from the cell‐cycle kinase cyclin‐dependent kinase 2 (CDK2) (FPKWKPGSLASHVKN) had no significant effect. The mutation of charged residues (H232→A, H234→A, D235→A) within a nearby minor insert sequence (HGHDNY, positions 232–237), or replacement of residues 234–237 with the corresponding residues from CDK2 (DSEI) also did not affect α/β subunit association and tetramerisation, but reduced enzyme thermal stability to more closely resemble the stability of the isolated α‐subunit. In addition, mutations within the minor insert caused approximately a threefold increase in the apparent Km for peptide substrate. The results indicate that the major and minor inserts are not essential for α/β subunit association, but the minor insert region influences substrate binding and thermal stability.
Redox‐Shift of the Pheromone‐Binding Protein in the Silkmoth Antheraea Polyphemus
Authors: Ziegelberger Gunde
Published: July 2010

In pheromone‐sensitive hairs of the male silkmoth Antheraea polyphemus, two electrophoretically distinct pheromone‐binding proteins (PBPs) are present. They indicate no amino acid sequence diversity according to peptide mapping, but differ in their redox state, as shown by free‐sulfhydryl‐group‐specific cleavage at cysteine residues with 2‐nitro‐5‐thiocyanobenzoic acid. In kinetic studies, the pheromone was initially bound mainly by the reduced PBP but later by the oxidized PBP, where all six cysteine residues form disulfide bonds. This redox shift was observed only in the homogenate of isolated olfactory hairs, where proteins of the sensillum lymph and receptive dendrites are present. In control experiments with purified binding proteins, the proportion of pheromone bound to the oxidized PBP did not increase with increasing incubation time, suggesting that disulfide formation does not occur spontaneously but is mediated by the sensory hairs, possibly by interaction with the receptor cell membrane. These data suggest that arriving hydrophobic pheromone molecules are first bound by the reduced PBP and transported through the aqueous sensillum lymph towards the receptor molecules of the dendritic membrane. The oxidized complex might not be able to activate further receptors and, thus, effectively deactivate the pheromone molecules within the sensillum lymph.
Hepatic Fatty Acid Metabolism as a Determinant of Plasma and Liver Triacylglycerol Levels
Authors: Asiedu Daniel K. , Al‐Shurbaji Ayman , Rustan Arild C. , Björkhem Ingemar , Berglund Lars , Berge Rolf K.
Published: July 2010

To investigate the importance of factors influencing substrate availability for triacylglycerol biosynthesis on lipoprotein metabolism, the effects of two opposite‐acting sulphur‐substituted fatty acid analogues, tetradecylthioacetic acid and tetradecylthiopropionic acid were studied. Administration of tetradecylthioacetic acid to rats resulted in a reduction of plasma levels of triacylglycerols (44%) and cholesterol (26%). This was accompanied by a reduction in very‐low‐density lipoprotein (VLDL) triacylglycerols (48%), VLDL cholesterol (36%), low‐density lipoprotein (LDL) cholesterol (36%) and high‐density lipoprotein (HDL) triacylglycerols (50%), whereas HDL cholesterol levels did not change. Subsequently, the HDL/LDL‐cholesterol ratio increased by 40%. The cholesterol‐lowering effect was accompanied by a reduction in hydroxymethylglutaryl CoA (HMG‐CoA) reductase activity (37%). Both mitochondrial and peroxisomal fatty acid oxidation increased (1.7‐fold and 5.3‐fold, respectively). Furthermore, there was a significant negative correlation between plasma triacylglycerols and mitochondrial fatty acid oxidation. Hepatic triacylglycerol synthesis was retarded, as indicated by a decrease in VLDL triacylglycerol secretion (40%), and by a reduced liver triacylglycerol content (29%). The activities of lipoprotein lipase and hepatic lipase in post‐heparin plasma were not affected. Microsomal and cytosolic phosphatidate phosphohydrolase activities were inhibited (28% and 70%, respectively). Hepatic malonyl‐CoA levels decreased by 29% and the total activity of acetyl‐CoA carboxylase was reduced (23%). In hepatocytes treated with tetradecylthioacetic acid, mitochondrial fatty acid oxidation increased markedly (100%) and triacylglycerol secretion was reduced (40%). In tetradecylthiopropionic‐acid‐treated rats, a significant increase in both plasma and VLDL triacylglycerols was found (46% and 72%, respectively) but VLDL triacylglycerol secretion was unaffected. However, no effect on either plasma or lipoprotein cholesterol levels was seen. Mitochondrial fatty acid oxidation was decreased by 50% and hepatic triacylglycerol levels increased by 33%. In hepatocytes exposed to tetradecylthiopropionic acid, triacylglycerol synthesis increased (100%) while triacylglycerol secretion and fatty acid oxidation remained unaltered. The results illustrate that lipoprotein triacylglycerol levels can be modulated by changes in the availability of fatty acid substrate for triacylglycerol biosynthesis, mainly by affecting mitochondrial fatty acid oxidation. In addition, we demonstrate that suppression of rat hepatic HMG‐CoA reductase activity during treatment with tetradecylthioacetic acid may contribute to a cholesterol‐lowering effect.
[3H]7‐Azido‐4‐Isopropylacridone Labels Cys159 of the Bovine Mitochondrial ADP/ATP‐Carrier Protein
Authors: Oettmeier Walter , Masson Klaus , Kalinna Sabine
Published: July 2010

Acridones (9‐azaanthracen‐10‐ones), especially halogen‐substituted acridone–carboxylic acids are efficient inhibitors of [14C]ADP binding to the bovine mitochondrial ADP/ATP‐carrier protein. 7‐Iodoacridone‐4‐carboxylic acid displaces [14C]ADP in a competitive manner, which indicates that both compounds share an identical binding site. Upon ultraviolet illumination, 7‐azido‐4‐[1′,2′‐3H]isopropyl‐acridone binds covalently to the ADP/ATP‐carrier protein. By sequencing of its proteolytic fragments, radioactivity was detected in Cys159. This amino acid is located close to Lys162, a target of 2‐azido‐[α‐32P]ADP [Dalbon, P., Brandolin, G., Boulay, F., Hoppe, J. & Vignais, P. V. (1988) Biochemistry 27, 5141–5149].
Membrane Lipid Composition and Cell Size of Acholeplasma laidlawii Strain A are Strongly Influenced by Lipid Acyl Chain Length
Authors: Wieslander Åke , Nordström Susanne , Dahlqvist Anders , Rilfors Leif , Lindblom Göran
Published: July 2010

The small, cell‐wall‐less prokaryote Acholeplasma laidlawii strain A‐EF22 could grow with membrane lipids having an average acyl chain length C̄n varying over 14.5– almost 20 carbons by exogenous supplementation with selected fatty acids. For 16 < C̄n < 18, the cells grew with lipids containing 100% (mol/100 mol) monounsaturated acyl chains, whereas for C̄n < 16 and En C̄n >18, cell growth only occurred with gradually lower fractions of unsaturated chains C̄n was actively increased and decreased by chain elongation or de novo fatty acid synthesis upon incorporation of short‐chain and long‐chain fatty acids, respectively.

The membrane lipid composition was strongly affected by the acyl chain length and unsaturation, and the metabolic responses are readily explained as a regulation mechanism based on the established phase equilibria of the individual lipids in the A. laidlawii membrane. Monoglucosyldiacylglycerol (Glc‐acyl2Gro) was the dominating lipid with short chains but the fraction of this lipid decreased with increasing C̄n correlating with the decreasing lamellar to nonlamellar phase transition temperatures for this lipid. The fractions of diglucosyldiacylglycerol (Glc2‐acyl2Gro) and phosphatidylglycerol (PtdGro), forming lamellar phases only, increased with increasing C̄n over the entire chain‐length interval. A weaker correlation was usually observed between the relative amount of a lipid and the extent of chain unsaturation; however, the fractions of Glc2‐acyl2Gro and PtdGro increased clearly with an increasing degree of unsaturation. Moreover, the synthesis of the nonbilayer‐forming lipids acyl2Gro and monoacyl‐Glc‐acyl2Gro was strongly stimulated by a high degree of chain saturation. Concomitantly, the phase equilibria of Glc‐acyl2Gro are shifted towards lamellar phases at the growth temperature. The fraction of the three potentially nonbilayer‐forming lipids varied over 10–80% (mol/100 mol) total lipids as a function of the acyl chain composition. The combined molar fractions of the three phospholipids increased strongly with chain unsaturation. However, the fraction of phosphate moieties in the different lipids was constant over the entire chain‐length interval. It is concluded that the regulation of the membrane lipid composition aims at maintaining similar phase equilibria and surface charge densities of the lipid bilayer.

The size of A. laidlawii cells was changed in a systematic manner and correlated qualitatively with the packing properties of the lipids. Cell diameters were increased by an increase in acyl chain length and saturation, and was affected by additives such an n‐dodecane and acyl2Gro.
Insulin‐Induced Proteolysis of the Insulin Receptor α‐Subunit from Rat Liver does not Occur in vivo but is Prevented in vitro by Blood Serum Proteinase Inhibitors
Authors: Sánchez‐Casas Paloma , Yusta Bernardo , Blázquez Enrique
Published: July 2010

Insulin binding to rat liver plasma membranes promotes the action of a plasma membrane proteinase that degrades the 135–kDa α‐subunit of the insulin receptor to a 120–kDa product. It has been proposed that this proteolysis may be the initial step leading to down‐regulation of the hepatic insulin receptor [Lipson, K. E., Kolhatkar, A. A. & Donner, D. B. (1988) J. Biol. Chem. 263, 10495–10501]. Our results confirm that liver plasma membrane fractions from intact or perfused rat liver and from isolated rat hepatocytes do have a proteinase that degrades the α‐subunit of the insulin receptor, whose activity increases significantly in the presence of insulin. In contrast to these in vitro results, plasma membranes and Golgi fractions isolated from liver at different times after a single intravenous injection of insulin to rats only contained intact α‐subunits of the insulin receptor. Insulin administration was associated with a rapid and reversible translocation of insulin receptors from the plasma membranes to Golgi fractions but did not affect their total numbers or the receptor half‐life. Incubation of rat liver plasma membranes with human, bovine or rat blood sera totally blocked α‐subunit proteolysis both in the absence or presence of insulin. The three major serum proteinase inhibitors, α1‐antitrypsin, α2‐macroglobulin and antithrombin III, blocked α‐subunit proteolysis of the insulin receptor to a varying extent. α1‐antitrypsin exhibited the highest potency, decreasing the amount of 120–kDa product by 70%. When both α1‐antitrypsin and antithrombin III were present, inhibition increased to 85%. Thus, the absence of proteolysis of the hepatic insulin receptor in vivo could be mostly accounted for by the antiproteolytic activity of blood serum. These findings suggest that insulin receptor α‐subunit proteolysis does not represent a mechanism involved in the down‐regulation of the insulin receptor in liver.
Molecular and Biochemical Characterization of aPlasmodium FalciparumCyclophilin Containing a Cleavable Signal Sequence
Authors: Hirtzlin Jocelyne , Färber Petra M. , Franklin Richard M. , Bell Angus
Published: July 2010

The immunosuppressive drug cyclosporin A (CsA) inhibits the growth of malaria parasitesin vitroandin vivo.Cyclosporin A exerts its immunosuppressive effect in T lymphocytes by binding to cyclophilin (CyP), a peptidylprolylcis‐transisomerase (PPIase). It is believed that the cyclosporin/falciparum complex inhibits a Ca2+‐activated protein phosphatase, calcineurin, involved in T‐cell activation. A cDNA encoding a cyclophilin of the human malaria parasitePlasmodium falciparumhas been isolated as a step in the elucidation of the mechanism of antimalarial action of CsA. This cDNA, termed PfCyP, encodes a protein of 195 amino acids which has highest similarity with theCandida albicans(73.1%) and theDrosophila melanogaster(73.1%) cytoplasmic cyclophilins. A Northern blot reveals an approximately 900–bp nucleotide transcript that is consistent with the predicted size of the encoded polypeptide. The predicted PfCyP protein has a putative endoplasmic‐reticulum‐directed signal sequence at its N‐terminus and two potential N‐linked glycosylation sites. Expression of PfCyP RNA in anin vitrotranslation/translocation system reveals that the PfCyP protein is translocated across microsomes, that the signal peptide is cleaved and that the PfCyP protein is glycosylated at two sites. The PfCyP cDNA open reading frame coding for the predicted mature protein has been expressed inEscherichia coli.The purified recombinant protein is an active PPIase (kcat/Km= 2.3×106s‐1M‐1); this enzymic activity is inhibited by CsA (IC50= 10 nM). The PfCyP protein has thus the same sensitivity to CsA as the PPIase activity associated withP. falciparumextracts [Bell, A. et al. (1994)Biochem. Pharmacol. 48, 495–503] suggesting that PfCyP may be responsible for the PPIase activity in those extracts. If different cyclophilins exist inP. falciparum, we conclude that either the PfCyP protein is the major cyclophilin detected in the parasite or that there are other cyclophilins with similar susceptibilities to CsA.