bluuebunny/crossref_metadata_2025 huggingface dataset:
Antimycin inhibition of the cytochrome bd complex from Azotobacter vinelandii indicates the presence of a branched electron transfer pathway for the oxidation of ubiquinol
Authors: Jünemann Susanne , Wrigglesworth John M.
Published: July 2002

Antimycin A and UHBDT inhibit the activity of the purified cytochrome bd complex from Azotobacter vinelandii. Inhibition of activity is non‐competitive and antimycin A binding induces a shift to the red in the spectrum of a b‐type haem. No inhibitory effects were seen with myxothiazol. Steady‐state experiments indicate that the site of inhibition for antimycin A lies on the low‐potential side of haem b
558. In the presence of antimycin A at concentrations sufficient to inhibit respiration, some direct electron transfer from ubiquinol‐1 to haem b
595 and haem d still occurs. The results are consistent with a branched electron transfer pathway from ubiquinol to the oxygen reduction site.
Effect of phosphotyrosine phosphatase over‐expression on glutathione metabolism in normal and oncogene‐transformed cells
Authors: Marraccini Patrizia , Iantomasi Teresa , Rigacci Stefania , Pacini Stefania , Ruggiero Marco , Vincenzini Maria Teresa , Ramponi Giampietro
Published: July 2002

We measured the level of reduced glutathione (GSH) and oxidized glutathione (GSSG) in normal and oncogene‐transformed NIH/3T3 fibroblasts and 32D hematopoietic cells. NIH/3T3 cells transformed by the activated oncogenes erbB, src, and raf, showed increased levels of GSH with concomitant alterations in the levels of GSH‐related enzymes. Transfection and over‐expression of a synthetic gene coding for a phosphotyrosine protein phosphatase (PTPase), which inhibited the proliferation of normal and transformed NIH/3T3 cells, was accompanied by a decrease in GSH levels in normal and erbB‐transformed fibroblasts, and by an increase in src and raf transformants. Among GSH‐related enzymes, only γ‐glutamylcysteine synthetase was altered in normal and erbB‐transformed NIH/3T3 fibroblasts following PTPase transfection. Therefore, tyrosine phosphorylation could be selectively involved in the regulation of GSH metabolism in normal and oncogene‐transformed NIH/3T3 fibroblasts, possibly by a dual‐type effect on receptor/oncoprotein‐mediated mitogenic signal transduction. However, no relationship was observed between the GSH and PTPase effect on cell growth, either after oncogene transfection or PTPase transfection. Moreover, the changes in GSH metabolism were specifically related to cell lineage. In fact GSH and related enzymes did not change in 32D hematopoietic cells transformed by the same activated erbB oncogene and in those ‐ normal or transformed over‐expressing the PTPase: in these cells also, over‐expression of the PTPase gene was not accompanied by growth inhibition.
Combination of the electrogenic ionophores, valinomycin and CCCP, can lead to non‐electrogenic K+/H+ exchange on bilayer lipid membranes
Authors: Orlov Victor N. , Antonenko Yuri N. , Bulychev Alexander A. , Yaguzhinsky Lev S.
Published: July 2002

The method of pH shift measuring by means of a pH microelectrode was applied to measure hydrogen ion fluxes across a planar bilayer lipid membrane (BLM) in the presence of the potassium ion ionophore, valinomycin, and a protonophore, carbonylcyanide m‐chlorophenylhydrazone (CCCP), under conditions of the voltage clamp. The voltage dependence of the flux was determined to be in the range of ± 150 mV under the conditions of both symmetrical KCl as well as a KCl gradient across the BLM. Surprisingly, at a clamped zero voltage on BLM a significant hydrogen ion flux was observed in the presence of a KCL gradient and both valinomycin and CCCP. This finding was interpreted as a result of induction of non‐electrogenic K+/H+ exchange in the presence of valinomycin and CCCP, presumably through the formation of electrically neutral complexes of these two ionophores and K+ (H+) ions: valinomycin—K+—CCCP− and/or possibly valinomycin—CCCP−—H+.
Post‐transcriptional repression of thymidine kinase expression during cell cycle and growth stimulation
Authors: Mikulits Wolfgang , Müllner Ernst W.
Published: July 2002

In vertebrates, endogenous thymidine kinase (TK) gene expression is strictly growth‐dependent. Here we report that in continuously cycling Ltk− mouse fibroblasts, stably transfected with a vector expressing human TK cDNA from a constitutive promoter, enzyme activity rises 8‐fold at the G1/S phase transition and declines again in G2. The mechanism did not involve changes in protein stability. When hTK was put under the control of a hormone‐inducible promoter, production of high mRNA levels following addition of dexamethasone did not result in any enzyme activity in resting NIH‐3T3tk− cells. After growth stimulation with serum, TK activity rose together with the onset of DNA synthesis only in the simultaneous presence of the hormone.
Stability and solvation of Thr/Ser to Ala and Gly mutations at the N‐cap of α‐helices
Authors: Yu Wai Chen , Fersht Alan R.
Published: July 2002

The solvation of polar groups at the N‐terminal end of α‐helices was studied by comparing the crystal structures of T4 lysozyme, barley chymotrypsin inhibitor 2 (CI2), barnase and their respective N‐cap mutants. Whether or not the N3 residue is solvated on mutating the N‐cap Thr/Ser to Ala or Gly appears to be related to the identities and the side‐chain conformations of the N2 and N3 residues. When these two residues are alanines, as is in the pseudo‐wild‐type CI2 (E33A/E34A), the main‐chain NH at the N3 position is exposed to the solvent and can be solvated. If the N2 residue is an Asp or a Glu, it is more likely that the side‐chain of these residues will form a surrogate N‐cap with the amide NH at N3 to compensate for the lost ‐OH group. In this case, no additional solvation will be observed. In general, Gly can be more stable than Ala at the N‐cap because its small side‐chain allows nearby polar groups to form hydrogen bonds with optimal geometry with solvent molecules or other polar groups.
Expression of the barley psbA gene in Escherichia coli yields a functional in vitro photosystem II protein D1
Authors: Efimov Vladimir A. , Fradkov Arkady F. , Raskind Alexander B. , Khristin Michael S. , Klimov Vyacheslav V. , Chakhmakhcheva Oksana G.
Published: July 2002

The barley chloroplast psbA gene encoding D1 protein, one of the main photosystem II components, has been over‐expressed in E. coli cells. The existance of two in vivo expression products, a protein with M
r about 33.5 kDa, corresponding to the full‐length precursor of the 32 kDa D1 mature form, and a truncated 29 kDa polypeptide was revealed. A modified D1 protein containing six histidine residues at the carboxy‐terminus was also obtained. After isolation and renaturation, the ability of the recombinant D1 protein to bind atrazine and pigments from barley thylakoids was demonstrated.
Possible role of the transcription factor interferon regulatory factor 1 (IRF‐1) in the regulation of ornithine decarboxylase (ODC) gene expression during IFNγ macrophage activation
Authors: Manzella Livia , Giuffrida Maria Assunta , Pilastro Maria Rosaria , Girlando Giuseppe , Picardi Giuseppina , Malaguarnera Lucia , Messina Angelo
Published: July 2002

In this report we discuss the role of interferon regulatory factor 1 (IRF‐1) in the regulation of ornithine decarboxylase (ODC) transcription during IFNγ human macrophage activation. We show that a binding sequence for the transcription factor IRF‐1 is contained in the first intron of the human ODC gene (from nt +2711 to nt +2722) we demonstrate that the level of expression of IRF‐1 increases in human macrophages and in the human promonocytic cell line, U937, previously differentiated in monocytes/macrophages by phorbol myristate acetate (PMA), after 2 h of IFNγ stimulation. We also show that the hamster tk‐ts13 cell line, stably transfected with the IRF‐1 cDNA, over‐expresses ODC. In addition, a specific complex was detected, by gel‐shift assay after incubating a 20 bp double‐stranded oligomer containing the binding sequence for IRF‐1 with nuclear proteins extracted from human macrophages and from (PMA‐differentiated) U937 cells stimulated with IFNγ for 2 h.
Antisense oligonucleotide complementary to endogenous retroviral MCF env gene inhibits both BFU‐E and CFU‐S colony formation in mice
Authors: Chernukhin Igor V. , Khaldoyanidi Sophia K. , Dikovskaya Dina V. , Svinarchuk Feodor P. , Vlasov Valentin V. , Gaidul Konstantin V.
Published: July 2002

A possible biologic activity of endogenously expressed env sequence of retroviral mink cell focus‐forming virus (MCF) genome for hematopoietic colony formation was studied in mice. Antisense 20‐mer complementary to MCF env sequence was used to detect the result of blockage of this gene translation on the potency of marrow cells to form colonies of erythroid (BFU‐E), myeloid granulocyte‐macrophage (CFU‐GM), and stem cell (day 11 CFU‐S) hematopoietic compartments. A large relative decrease in BFU‐E number was found in bone marrow cell cultures preincubated with antisense oligonucleotide during 4 h, whereas CFU‐GM colonies remained unaffected. A marked reduction of CFU‐S colony formation was also registered under antisense oligomer influence. Following a decreased proliferation of erythroid progenitors, we suggest the mechanism by which antisense oligonucleotide could cause the loss of colony formation. Taken together, these data allow to propose that the expression of this gene is naturally significant for hematopoietic progenitor activity exerting some property of env gene products to regulate the growth of erythroid and multilineage hematopoietic precursors.
Identification of spliced RNA species of Drosophila melanogaster gypsy retrotransposon New evidence for retroviral nature of the gypsy element
Authors: Avedisov Sergei N. , Ilyin Yurii V.
Published: July 2002

We have identified a novel RNA species of Drosophila melanogaster gypsy retrotransposon that is ca. 2 kb in length and corresponds to the third open reading frame (ORF3) of the gypsy element. This RNA is generated by splicing of the primary gypsy transcript, as is the case for retroviral env gene expression. Therefore, the striking resemblance between gypsy and retroviruses has now been extended by this study to the expression strategies of these retroelements. The primary structure of spliced RNA was determined, and its analysis shows that both gypsy subfamilies (6K and 7K) apparently are able to encode functionally active ORF3 translation products.
The nucleotide sequence of a complementary DNA encoding Flaveria bidentis carbonic anhydrase
Authors: Cavallaro Antonino , Ludwig Martha , Burnell James
Published: July 2002

We have isolated and characterised a cDNA clone encoding the cytosolic form of carbonic anhydrase in the leaves of Flaveria bidentis, a C4 dicotyledonous plant. The deduced amino acid sequence is similar to the carbonic anhydrase found in the chloroplasts of C3 dicotyledonous plants. Western blot analysis of crude leaf extracts of F. bidentis indicates that the leader sequence (equivalent to the transit peptide of the chloroplastic form of CA found in C3 plants) is not removed following translation of mRNA.
Epidermal growth factor induces protein tyrosine phosphorylation and association of p190 with ras‐GTP‐ase activating protein in Caco‐2 cells
Authors: Auricchio Alberto , Domenico Marina di , Castoria Gabriella , Bilancio Antonio , Migliaccio Antimo
Published: July 2002

Epidermal growth factor (EGF) modulates several functions of human enterocytes. We report that this growth factor induces strong tyrosine phosphorylation stimulation of its receptor and several putative substrates of the receptor intrinsic kinase including c‐erb B2 in proliferating human colon carcinoma cells (Caco‐2). In addition EGF induces stable association of the GTP‐ase activating protein of p21ras to the p190 protein and to a 62 mol.wt. tyrosine‐phosphorylated protein. This association is probably consequent to EGF stimulation of protein tyrosine phosphorylation and could coordinate progression through cell cycle with polarity, cell—cell interactions and cell mobility.
A unique biosynthetic pathway for gangliosides exists in Xenopus laevis oocytes
Authors: Hidari Kazuya I.-P.Jwa , Nagai Yoshitaka , Sanai Yutaka
Published: July 2002

It was previously reported that monosialosylgangliopentaosyl ceramide (Ga1NAc‐GM1b) was a major ganglioside in Xenopus laevis oocytes. Here we determined biosynthetic pathways for the ganglioside by detailed measurements of glycosyltransferase activities. CMP‐NeuAc:asialo‐GM1 α2–3 sialyltransferase (α2–3 ST) and UDP‐Ga1NAc:GM1b β1–4 N‐acetylgalactosaminyltransferase (β1–4 Ga1NAcT) exhibited much higher activity than CMP‐NeuAc:Ga1NAc‐GA1 α2–3 ST and UDP‐Ga1NAc:asialo‐GM1 β1–4 Ga1NAcT, respectively. These observations indicated the existence of a unique biosynthetic pathway in the oocytes as follows; asialo‐GM1 → GM1b → Ga1NAc‐GM1b.
Effect of substitution of hemagglutinin‐neuraminidase with influenza hemagglutinin on Sendai virus F protein mediated membrane fusion
Authors: Bagai Sangeeta , Sarkar Debi P.
Published: July 2002

Recombinant virosomes containing fusion protein (F) of Sendai virus and the envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA) of influenza virus within the same membrane were prepared. Such hybrid vesicles were found to hemolyse red blood cells both at pH 7.4 and pH 5.0. Hemolysis induced by hybrid vesicles was much higher than seen with F‐virosomes in the presence of WGA, but was about two‐fold less than the hemolysis caused by F,HN‐virosomes. Reconstituted influenza virus envelopes and F‐virosomes failed to induce hemolysis at pH 7.4. Using a fluorescence probe‐based lipid mixing fusion assay, hybrid virosomes were found to fuse with cultured HeLa cells both at pH 7.4 as well as pH 5.0. The data indicate that the presence of Sendai virus HN protein in the virosomal membrane is not absolutely essential for the virosome‐cell fusion process.
Characterisation of a complementary DNA encoding a novel plant enzyme with sucrolytic activity
Authors: Machray Gordon C. , Burch L. , Hedley P.E. , Davies H.V , Waugh R.
Published: July 2002

The cloning of a 1332 bp cDNA from a potato (Solanum tuberosumL.) cv. Cara leaf cDNA expression library, using an antibody raised against a purified tuber protein preparation with sucrolytic activity, is described. The corresponding gene in potato is of low copy number, is expressed in a variety of tissues, and encodes a protein which includes several domains with similarity to database sequences, including ferredoxin from Clostridium pasteurianum. Expression of the cDNA in E. coli yields a fusion protein with sucrolytic activity.
Characterization of the promoters of the guinea pig neutrophil cationic peptide‐1 and ‐2 genes
Authors: Nagaoka Isao , Ishihara Noriko , Yamashita Tatsuhisa
Published: July 2002

Guinea pig neutrophils contain the antimicrobial cationic peptides GNCP‐1 and GNCP‐2 in the granules. To understand the regulation of the gene expression, the promoters for the GNCP‐1 and GNCP‐2 genes were characterized. Sequencing analysis of the genomic clones showed that the nucleotide sequences of the 5′‐upstream regions (1.7 kb) from exon 1 were homologous (90–93%) between the GNCP‐1 and GNCP‐2 genes. However, transient transfection assays using luciferase reporter gene constructs revealed that the promoter activity of GNCP‐1 was 2‐fold greater than that of GNCP‐2. Furthermore, DNase I footprint analysis demonstrated that three regions (I, II and III) were protected on the GNCP‐1 promoter, whereas only two protected regions (II and III) were identified on the GNCP‐2 promoter. Together these observations indicate that GNCP‐1 and GNCP‐2 are encoded by homologous genes, but the expression of the GNCP‐1 and GNCP‐2 genes is likely to be different at the level of transcription.
Residues responsible for distinct biological functions are characterized by different statistical features of sequential and spatial neighborhoods: A thermolysin example
Authors: Gabrielian A.E. , Kostrov S.A. , Kirpichnikov M.P.
Published: July 2002

An investigation of the functional topography of thermolysin was carried out using frequency analysis of its primary and tertiary structures. The statistical validity of this approach was estimated for the enzyme active site, the substrate‐binding pocket, the inter‐domain interface and calcium‐binding sites' predictions. We showed that frequency analysis of primary structure could be employed to predict the localization of contiguous parts of the inter‐domain interface. The same approach appears to be unsuitable to a search for conformation‐dependent enzyme active sites and substrate‐binding pockets. In contrast, frequency analysis of the spatial neighborhood is not effective for predicting the inter‐domain interface as distinct from the active site, substrate‐binding pocket and calcium‐binding sites. These differences should be taken into account when investigating and understanding protein structure—function relationships.
Induction of protein‐tyrosine phosphatase LC‐PTP by IL‐2 in human T cells
Authors: Adachi Masaaki , Sekiya Masuo , Ishino Masaho , Sasaki Hiroko , Hinoda Yuji , Imai Kohzoh , Yachi Akira
Published: July 2002

Tyrosine phosphorylation has been implicated in interleukin 2 (IL‐2)‐induced growth signaling and the phosphorylation levels are regulated by the balance of tyrosine kinase and tyrosine phosphatase activities. Here, we demonstrate the rapid activation of a leukocyte tyrosine phosphatase LC‐PTP (HePTP) gene expression by IL‐2 in an IL‐2 dependent human T cell ILT‐Mat. Accumulation of LC‐PTP mRNA appeared at l h and peaked at 6 h after IL‐2 stimulation, simultaneous with the Gl to early S phase, and the induction of LC‐PTP mRNA did not require protein synthesis. LC‐PTP protein increased approximately 6‐fold at 8 h after IL‐2 stimulation. Nuclear run‐on assays showed that the induction of LC‐PTP mRNA expression is mostly due to transcriptional activation. These data suggest that LC‐PTP is an early response gene and its protein seems to be a crucial molecule which regulates the tyrosine phosphorylation level during T cell proliferation.
Photoaffinity cross‐linking of F1ATPase from spinach chloroplasts by 3'‐arylazido‐β‐alanyl‐8‐azido ATP
Authors: Schäfer Hans-Jochen , Rathgeber Gabriele , Schuhen Achim , Berzborn Richard J.
Published: July 2002

UV irradiation of the ATPase (CF1) from spinach chloroplasts in the presence of 3'‐arylazido‐β‐alanyl‐8‐azido ATP (8,3'‐DiN3ATP) results in a nucleotide‐dependent inactivation of the enzyme and in a nucleotide‐dependent formation of α‐β cross‐links. The results demonstrate an interfacial localization of the nucleotide binding sites on CF1.
Noncanonical Oct‐sequences are targets for mouse Oct‐2B transcription factor
Authors: Stepchenko A.G.
Published: July 2002

We have suggested a random modification method for determining preferable binding sites of a DNA‐binding protein and applied this method to the Oct‐2B transcription factor. Our results indicate that the Oct‐2B protein interacts with canonical oct sequence ATGC/TAAAT and degenerated sequences which contain TAAT motif in the binding site. We have determined nucleotides in the binding sites, involved in the DNA‐protein interaction, and the equilibrium dissociation constants K
d for these sequences. These data show that a much greater number of potential targets for Oct proteins exist on DNA and changed our view on the gene expression regulation by this protein factor.
Crystallisation of a low molecular weight phosphotyrosine protein phosphatase from bovine liver
Authors: Su Xiao-Dong , Agango Elisha Gor , Taddei Niccolo' , Bucciantini Monica , Stefani Massimo , Ramponi Giampietro , Nordlund Pär
Published: July 2002

Single crystals of a low molecular weight phosphotyrosine protein phosphatase from bovine liver have been grown. The crystals belong to space group P212121, have cell dimensions a = 46.3 Å, b ‐ 62.2 Å, c = 62.7 Å and diffract to better than 2.0 Å resolution. The crystals are well suited for high resolution X‐ray studies.
Glucose transporter localization in rat skeletal muscle
Authors: Dudek Ronald W. , Dohm G.Lynis , Holman Geoffrey D. , Cushman S.W. , Wilson Cindy M.
Published: July 2002

Surface glucose transporters of intact muscles were photolabeled with the membrane impenneant ATB‐[2‐3H]BMPA reagent and localized by autoradiography. We found sparse labeling of the glucose transporters by ATB‐[2‐3H]BMPA on the sarcolemmal membrane around the muscle fiber. The majority of label was on the interior of the muscle fiber, at a discrete site which matched the distribution of AI junctions and which was presumed to be on the exterior surface of T‐tubules. The amount of photolabel on the T‐tubule was increased in response to insulin and wasblocked by cytochalasin B. These results support the concept that glucose transport may occur predominately across the T‐tubule membrane under basal and insulin‐stimulated conditions.
Ascorbic acid can either increase or decrease low density lipoprotein modification
Authors: Stait Suzanne E. , Leake David S.
Published: July 2002

In freshly prepared low density lipoprotein (LDL), ascorbate inhibited LDL oxidation by macrophages at the higher concentrations tested (60–100 μM). In contrast, with LDL that had been allowed to autoxidise in the refrigerator (3°C) for at least 10 weeks after isolation (mildly oxidised or minimally‐modified LDL), ascorbate did not inhibit the modification of LDL in the presence of macrophages. Ascorbate actually modified autoxidised LDL itself in the absence of macrophages to greatly increase its uptake by macrophages. The modification of autoxidised LDL by ascorbate increased the levels of thiobarbituric acid‐reactive substances in the medium and was completely inhibited by the antioxidant butylated hydroxytoluene. Thus the effects of ascorbate on unoxidized LDL can be very different to those on mildly oxidised LDL.
Inactivation of an NADPH‐dependent succinic semialdehyde reductase by o‐phthalaldehyde
Authors: Cho Sung-Woo , Hong Joung Woo , Lee Su Jin , Choi Soo Young
Published: July 2002

Incubation of an NADPH‐dependent succinic semialdehyde reductase from bovine brain with o‐phthalaldehyde resulted in a time‐dependent loss of enzyme activity. The inactivation followed pseudo first‐order kinetics with the second‐order rate constant of 28 M−1 s−1. The inactivation was prevented by preincubation of the enzyme with NADPH, but not by succinic semialdehyde. There was a linear relationship between isoindole formation and the loss of enzyme activity. Spectrophotometric studies indicated that complete inactivation of the enzyme resulted from the formation of one isoindole derivative per molecule of enzyme, which was formed from the reaction of cysteine and lysine residues with o‐phthalaldehyde at or near the enzyme active site.
Phospholipase D is involved in cytosolic phospholipase A2‐dependent selective release of arachidonic acid by fMLP‐stimulated rat neutrophils
Authors: Fujita Ken-ichi , Murakami Makoto , Yamashita Fumio , Amemiya Kouji , Kudo Ichiro
Published: July 2002

When rat polymorphonuclear neutrophils (PMN) were treated with N‐formyl‐Met‐Leu‐Phe (fMLP), the release of arachidonic acid in preference to other fatty acids was observed. Levels of arachidonic acid reached a plateau within 5 min, and were accompanied by an ∼ 4‐fold increase in in vitro phospholipase (PL) A2 and PLD activities in PMN lysates. Treatment of PMN with ethanol (an inhibitor of PLD‐mediated phosphatidic acid formation), propranolol (a phosphatidic acid phosphatase inhibitor), or 4‐bromophenacylbromide (a PLA2 inhibitor), each suppressed fMLP‐stimulated arachidonate release. Treatment with RHC‐80267 (a diacylglycerol lipase inhibitor), however, had no such effect. The cytosolic PLA2 (cPLA2) inhibitor, arachidonoyl trifluoromethyl ketone, suppressed PLA2 activity in PMN homogenates and arachidonate release by fMLP‐treated PMN. These results suggest that fMLP‐elicited arachidonate release is mediated by cPLA2 but not diacylglycerol lipase, and that the activation of cPLA2 is downstream of the PLD‐dependent signaling pathway.
Response of clomipramine in sexual addiction
Authors: Azhar MZ , Varma SL
Published: July 2002

Two cases of sexual addiction for more than five years treated with psychological treatment and clomipramine showed marked improvement after six to nine months. It is postulated that these disorders could be variants of obsessive compulsive disorder.
Cyclic alternating pattern (CAP) rate in the polygraphic investigation of classic and novel hypnotic drugs
Authors: Parrino L , Fioriti G , Terzano MG
Published: July 2002

The difficulty to recruit homogeneous samples of insomniacs requires alternative approaches for sleep studies. Acoustic perturbation in healthy volunteers made it possible to determine an experimental model of acute situational insomnia in order to investigate the effects of classic and novel hypnotic compounds. Unfortunately, the traditional scoring parameters of sleep are inadequate to provide reliable information for defining the neurophysiological bases of insomnia and for evaluating the efficacy of hypnotic drugs. Recent studies on the microstructure of sleep have permitted to identify a specific EEG feature, the cyclic alternating pattern (CAP), correlated with the subjective appreciation of sleep quality. Comparing placebo, zolpidem, zopiclone, lorazepam and triazolam, given at equivalent therapeutic doses in middle-aged healthy volunteer subjects under basal conditions and under acute situational insomnia, provided non-significant information when using classical sleep parameters whereas CAP rate (the percentage ratio of CAP time to non-REM sleep time) permitted to discriminate the basal nights from the perturbed nights and the drug nights from the placebo nights. These data have been confirmed in clinical studies using zolpidem versus placebo in chronic insomniacs.
14‐3‐3τ associates with a translational control factor FKBP12‐rapamycin‐associated protein in T‐cells after stimulation by pervanadate
Authors: Mori Hiroshi , Inoue Masahiro , Yano Mihiro , Wakabayashi Hideki , Kido Hiroshi
Published: July 2002

Proteins of the 14‐3‐3 family can associate with and/or modulate the activities of a variety of proteins, such as protooncogene and oncogene products, Cdc25 phosphatases and phosphatidylinositol 3‐kinase, and thus are implicated in regulation of signaling pathways and the cell cycle. We report here that treatment of Jurkat T‐cells with an inhibitor of protein tyrosine phosphatase, pervanadate, induces the association of 14‐3‐3τ with a translational control factor, FKBP12‐rapamycin‐associated protein (FRAP), with significant latter's autophosphorylation. Coimmunoprecipitation of various mutants of FRAP coexpressed with 14‐3‐3τ in COS‐7 cells revealed that 14‐3‐3τ binds to the C‐terminal side of FRAP at unknown site(s) different from the predicted binding motifs to date.
Different lumen‐targeting pathways for nuclear‐encoded versus cyanobacterial/plastid‐encoded Hcf136 proteins
Authors: Hynds Peter J. , Plücken Henning , Westhoff Peter , Robinson Colin
Published: July 2002

Lumenal proteins are transported across the thylakoid membrane by two very different pathways: Sec‐dependent or twin‐arginine translocase (Tat)‐dependent, where the substrate protein can be transported in a folded state. We present the first evidence that a given protein can be targeted by different pathways in different organisms. Arabidopsis Hcf136 is targeted exclusively by the Tat pathway in pea chloroplasts and no Sec‐dependent transport is evident even when the twin‐arginine is replaced by twin‐lysine. However, twin‐arginine motifs are absent from the presequences of Hcf136 proteins encoded by plastid or cyanobacterial genomes, strongly implying translocation by another pathway (presumably Sec). We suggest that the Hcf136 protein was transferred to the Tat pathway when the gene became incorporated into the nuclear genome, possibly due to the tighter folding associated with the more involved, post‐translational targeting pathway.
Genomic structure and promoter analysis of putative mouse acetyl‐CoA transporter gene
Authors: Bora Roop Singh , Ichikawa Shinichi , Kanamori Akiko , Hirabayashi Yoshio
Published: July 2002

The acetyl‐CoA transporter gene (Acatn) encodes a hydrophobic, multitransmembrane protein that is involved in the process of O‐acetylation of sialic acid residues on gangliosides. O‐Acetylated gangliosides have been found to play important roles in tissue development and organization during early embryonic stages. We have cloned the gene for mouse acetyl‐CoA transporter. The gene spans approximately 20 kb and is composed of seven exons and six introns. A single transcription initiation site, 371 bp upstream of the ATG start codon, was identified. The promoter region was found to lack a TATA box. However, several potential transcription factor binding motifs such as AP1, AP2, C/EBPα, C/EBPβ, HSF, GATA2 and MZF1 were identified in the promoter region.
Microtubule binding of the Drosophila DMAP‐85 protein is regulated by phosphorylation in vitro
Authors: Cambiazo Verónica , Logarinho Elsa , Pottstock Hans , Sunkel Claudio E
Published: July 2002

The phosphorylation of microtubule‐associated proteins (MAPs) is thought to be a key factor in the regulation of microtubule (MT) stability. Previously we isolated DMAP‐85, a Drosophila MAP shown to be associated with stable MTs. In this work we show that DMAP‐85 phosphorylated in cell‐free early embryo extracts is released from MTs. MPM‐2 antibodies recognize the phosphorylated protein. In vitro, DMAP‐85 can be phosphorylated by the mitotic kinase Polo affecting its binding to MTs and creating MPM‐2 epitopes on the protein. The results suggest that phosphorylation of DMAP‐85 might affect its MT stabilizing activity during early mitotic cycles.
Glycosylation of the calcitonin receptor‐like receptor at Asn60 or Asn112 is important for cell surface expression
Authors: Bühlmann Nicole , Aldecoa Amaya , Leuthäuser Kerstin , Gujer Remo , Muff Roman , Fischer Jan A , Born Walter
Published: July 2002

The human calcitonin (CT) receptor‐like receptor (hCRLR) of the B family of G protein‐coupled receptors is N‐glycosylated and associates with receptor‐activity‐modifying proteins for functional interaction with CT gene‐related peptide (CGRP) or adrenomedullin (ADM), respectively. Three putative N‐glycosylation sites Asn60, Asn112 and Asn117 are present in the amino‐terminal extracellular domain of the hCRLR. Tunicamycin dose‐dependently inhibited the glycosylation of a myc‐tagged hCRLR and in parallel specific [125I]CGRP and ‐ADM binding. Similarly, the double mutant myc‐hCRLR(N60,112T) exhibited minimal N‐glycosidase F sensitive glycosylation, presumably at the third Asn117, and the cell surface expression and specific radioligand binding were impaired. Substitution of the Asn117 by Thr abolished CGRP and ADM binding in the face of intact N‐glycosylation and cell surface expression.
Harpin, a hypersensitive response elicitor from Erwinia amylovora, regulates ion channel activities in Arabidopsis thaliana suspension cells
Authors: El-Maarouf Hayat , Barny Marie Anne , Rona Jean Pierre , Bouteau François
Published: July 2002

HrpN, the hypersensitive response elicitor from Erwinia amylovora, stimulated K+ outward rectifying currents in Arabidopsis thaliana suspension cells. It also decreased anion currents. These data demonstrate the ability of harpin to regulate different plasma membrane ion channels, putative components of signal transduction chains leading to defense responses and programmed cell death.
Structure requirement and identification of a cryptic cleavage site in the mitochondrial processing of a plant F1‐ATPase β‐subunit presequence
Authors: Duby Geoffrey , Degand Hervé , Boutry Marc
Published: July 2002

We sought to determine the structural features involved in the processing of the mitochondrial F1‐ATPase β‐subunit (F1β) presequence (54 residues) from Nicotiana plumbaginifolia. The cleavage efficiency of F1β presequence mutants linked to the green fluorescent protein (GFP) was evaluated in vivo in tobacco by in situ microscopy and Western blotting. The residue at position −1 (Tyr) was required to be an aromatic residue and the residue at position +2 (Thr) was found to be important for F1β processing, while, unexpectedly, changing the distal (Arg‐15) and proximal (Arg‐5) arginine residues did not strongly reduce processing. In addition, results also supported the requirement of a helical structure around the cleavage position. Sequencing of the mature form of a precursor containing the first 30 residues of the F1β presequence linked to GFP revealed the presence of a cryptic cleavage site between residues 26 and 27, which showed the features of a classical mitochondrial processing site, suggesting dual processing of the F1β presequence. In vitro processing confirmed these data and showed that processing was sensitive to o‐phenanthroline, thus catalyzed by mitochondrial processing peptidase.
Residue Glu‐91 of Chlamydomonas reinhardtii ferredoxin is essential for electron transfer to ferredoxin‐thioredoxin reductase
Authors: Jacquot Jean-Pierre , Stein Mariana , Suzuki Akira , Liottet Sandrine , Sandoz Gabriel , Miginiac-Maslow Myroslawa
Published: July 2002

The [2Fe‐2S] soluble ferredoxin from Chlamydomonas reinhardtii was mutated by site directed mutagenesis, using PCR and the expression plasmid pET‐Fd as a template. The recombinant mutated proteins were purified to homogeneity and tested in the activation of NADP‐malate dehydrogenase, a light dependent reaction in which ferredoxin thioredoxin reductase (FTR) and thioredoxin are involved. The mutation of residue Glu‐91 (E92 in spinach, E94 in Anabaena) alone, either to Gln (E91Q) or to Lys (E91K), was found to completely abolish the reaction of the enzyme light activation. On the other hand, the mutants (E92Q) or (E92K) were as efficient as the wild type ferredoxin in this reaction whereas the double mutants (E91Q/E92Q) or (E91K/E92K) had no activity. In addition, a triple mutant (D25A/E28Q/E29Q) was also found to be inactive for this redox dependent light activation. All these mutations had much weaker effects on the ferredoxin/ferredoxin NADP reductase interaction as measured by the cytochrome c reduction assay. These results indicate that there is a recognition site for FTR in the C terminus part of ferredoxin, but also that a core of negatively charged residues in the α1 helix of ferredoxin might be important in the general process of light activation.
Monoclonal antibodies that distinguish between free and complexed heterotrimeric G protein β subunits
Authors: Rehm Armin , Ploegh Hidde L
Published: July 2002

Heterotrimeric G proteins were purified from bovine brain by immunoaffinity chromatography on immobilized anti G protein monoclonal antibody 3C2. Release of βγ subunits was effectuated by exposure of immobilized trimeric G proteins to MgAlF4. The resultant βγ subunits were pure and biologically active. Following immunization of mice with purified βγ subunits we obtained monoclonal anti β antibodies showing broad species cross‐reactivity. Characterization of the epitope recognized by one such monoclonal antibody, ARC9, indicated involvement of the extreme COOH‐terminus, as assessed by its reactivity on β subunits lacking the COOH‐terminal 15 residues, obtained by in vitro translation. Although we used native βγ subunits as immunogen, all monoclonal antibodies obtained failed to recognize assembled βγ subunits, and were specific for free β subunits. This property is useful in characterizing the assembly of G proteins from their subunits in living cells.
Molecular cloning and immunological characterisation of Cyn d 7, a novel calcium‐binding allergen from Bermuda grass pollen1
Authors: Suphioglu Cenk , Ferreira Fatima , Knox R.Bruce
Published: July 2002

A cDNA coding for a newly identified Bermuda grass pollen allergen, Cyn d 7, with significant sequence similarity to Ca2+‐binding proteins, was isolated from a cDNA expression library using serum IgE from an allergic individual. The deduced amino acid sequence of Cyn d 7 contained two typical Ca2+‐binding sites (EF hand domains). Depletion of Ca2+ with EGTA led to a loss of IgE‐binding capacity of rCyn d 7. A synthetic peptide based on domain II showed high IgE reactivity. Cyn d 7 therefore represents a grass pollen allergen that belongs to a novel class of Ca2+‐binding proteins.
Cca3, the mRNA level of which transiently decreases before initiation of DNA synthesis in regenerating rat liver cells1
Authors: Hayashi Yasuyuki , Ichinose Masutaro , Yuasa Hirofumi , Tatematsu Masae , Ishibashi Masahide
Published: July 2002

Kinetics of cca1, cca2, cca3 and rat gas1 mRNA levels were compared with those of DNA synthesis level in regenerating rat liver cells after partial hepatectomy. A transient decrease of cca3 mRNA level and an increase of rat gas1 mRNA level were observed before initiation of DNA synthesis, followed by a rapid decrease of rat gas1 mRNA level. By molecular cloning and nucleotide sequencing, cca3 cDNA was found to consist of 4514 nucleotides with a large open reading frame of 3027 nucleotides, encoding a protein of 1009 amino acids with three copies of an ankyrin repeat‐like sequence.
Recombinant human O6‐alkylguanine‐DNA alkyltransferase induces conformational change in bound DNA
Authors: Federwisch Matthias , Hassiepen Ulrich , Bender Kirsten , Rajewsky Manfred F , Wollmer Axel
Published: July 2002

Circular dichroism, and steady‐state and time‐resolved fluorescence spectroscopy were used to compare the native recombinant human DNA repair protein O
6‐alkylguanine‐DNA alkyltransferase (AGT) with AGT bound to ds‐DNA. Contrary to fluorescence, analysis of the far‐UV CD spectra indicated a conformational change of AGT upon binding to DNA: its α‐helical content is increased by ∼12%. Analysis of near‐UV CD spectra revealed that DNA was also affected, probably being separated into single strands locally.
The Rhodobacter sphaeroides flagellar motor is a variable‐speed rotor
Authors: Packer Helen L , Lawther Helen , Armitage Judith P
Published: July 2002

The rotation rate of the unidirectional stop/start motor of Rhodobacter sphaeroides was investigated using computerised motion analysis of tethered cells. The R. sphaeroides motor was found to have a variable rotation rate compared to the virtually constant‐speed motor of wild‐type and CheR mutant (smooth swimming) Escherichia coli. In addition, the dynamics of the R. sphaeroides motor during stopping was analysed with no consistent correlation behaviour. The motor could go from full rotation to stop, or stop to full rotation within one video frame, i.e. 0.02 s, but it could also slow down into a stop or restart slowly, taking up to 0.25 s. The R. sphaeroides motor under chemokinetic stimulation was also analysed and was found to show increased torque generation and reduced variation in rotation rate.
Difference between PA700‐like proteasome activator complex and the regulatory complex dissociated from the 26S proteasome implies the involvement of modulating factors in the 26S proteasome assembly
Authors: Sawada Hitoshi , Akaishi Takahiro , Katsu Masataka , Yokosawa Hideyoshi
Published: July 2002

The PA700‐like proteasome activator complex was highly purified from porcine erythrocytes, and its properties were compared with those of the regulatory complex disassembled from the purified 26S proteasome. The molecular mass of the PA700‐like complex, which comprises 25–110‐kDa subunits, was estimated to be 800 kDa by Superose 6 gel filtration. This complex showed neither ATPase activity nor peptidase activity toward Suc–Leu–Leu–Val–Tyr–MCA. Nevertheless, it was possible to make a high molecular mass complex from the purified PA700‐like complex by incubating with the 20S proteasome in the presence of ATP. In contrast, the regulatory complex dissociated from the 26S proteasome did not reconstitute a larger complex under the same conditions. The subunit composition of the PA700‐like complex was similar but not identical to that of the regulator complex dissociated from the 26S proteasome: the former complex had a 25‐kDa subunit which is absent in the latter, whereas the latter had two or three 43‐kDa subunits lacking in the former. These results indicate that the purified PA700‐like proteasome activator complex is structurally and functionally distinct from the regulatory complex dissociated from the 26S proteasome, implying the involvement of modulating factors in the 26S proteasome assembly.
Domain movement in rabbit muscle adenylate kinase might involve proline isomerization
Authors: Sheng X.R , Zhang H.J , Pan X.M , Li X.F , Zhou J.M
Published: July 2002

The fluorescence probe, 8‐anilino‐1‐naphthalenesulfonic acid (ANS), was used to monitor the induced‐fit conformational movement in rabbit muscle adenylate kinase. In 50 mM Tris‐HCl buffer (pH 8.1), the time course of ANS binding to rabbit muscle adenylate kinase is a biphasic process. The fast phase completes within the dead‐time of the stopped‐flow equipment used (about 15 ms), while the slow phase ends in about 10 minutes. In the presence of 2.0 μM peptidyl prolyl cis/trans‐isomerase, the rate constant of the slow phase reaction is accelerated about 2.4‐fold, suggesting that the domain movement during ANS binding to rabbit muscle adenylate kinase may involve proline isomerization. The activation energy of the slow phase was determined to be 74.6 kJ/mol, which is comparable to the activation energy of proline cis/trans‐isomerization (about 80 kJ/mol).
HIV protease substrate conformation: modulation by cyclophilin A
Authors: McCornack Melissa A , Kakalis Lazaros T , Caserta Carlo , Handschumacher Robert E , Armitage Ian M
Published: July 2002

Cyclophilin A (CyPA), a cytosolic peptidyl‐prolyl trans‐cis isomerase can accelerate the trans‐cis isomerization of Xxx‐Pro peptide bonds. One‐ and two‐dimensional 1H‐NMR spectroscopy were used to determine that the heptapeptide Ser‐Gln‐Asn‐Tyr‐Pro‐Ile‐Val, a model peptide of an HIV‐1 protease cleavage site in the gag polyprotein of HIV‐1, is a substrate for CyPA. Experiments revealed a slow exchange about the Tyr‐Pro peptide bond with 30±5% in the cis conformation (pH 1–9). While the interconversion rate is too slow to measure by kinetic NMR methods in the absence of CyPA, these methods, saturation transfer and NOE experiments, established that CyPA enhanced the rate of trans‐cis interconversion, a process inhibited by cyclosporin A (CsA). With a substrate:CyPA ratio of 40:1, an interconversion rate of 2.5 s−1 at 25°C was observed.
The transcriptional co‐activator proteins p300 and CBP stimulate adenovirus E1A conserved region 1 transactivation‐independent of a direct interaction
Authors: Mannervik Mattias , Akusjärvi Göran
Published: July 2002

p300 and CBP are two related transcriptional co‐activator proteins required by many cellular transcription factors for activity. The adenovirus E1A protein binds p300 and CBP through its amino‐terminus and conserved region (CR) 1. Fusing CR1 to a heterologous DNA‐binding domain creates a potent transcriptional activator, suggesting that CR1 might activate transcription by recruiting p300/CBP to the promoter. We show that both p300 and CBP enhances CR1‐dependent transactivation. However, this enhancement occurs independently of a direct interaction with E1A and does not correlate with the CR1 activator function.
Stoichiometry of 7‐ethoxycoumarin metabolism by cytochrome P450 2B1 wild‐type and five active‐site mutants
Authors: Fang Xiaojun , Kobayashi Yasuna , Halpert James R
Published: July 2002

Recombinant P450 2B1 wild‐type and the active‐site mutants I114V, F206L, V363A, V363L, and G478S were purified and studied. The efficiency of coupling of reducing equivalents to 7‐hydroxycoumarin formation was decreased for all the mutants except I114V. Uncoupling to H2O was increased for F206L, V363A, and G478S, decreased for V363L, and unchanged for I114V. Uncoupling to H2O2 was increased for V363L and decreased for I114V, F206L, and V363A. The findings from this study provide firm biochemical evidence that residues 206, 363, and 478 comprise part of the substrate binding site of P450 2B1.
Peptide binding and photo‐crosslinking to detergent solubilized and to reconstituted transporter associated with antigen processing (TAP)
Authors: Uebel Stephan , Plantinga Titia , Weber Peter J.A , Beck-Sickinger Annette G , Tampé Robert
Published: July 2002

The transporter associated with antigen processing (TAP) is essential for peptide loading onto major histocompatibility (MHC) class I molecules by translocating peptides into the endoplasmic reticulum. We have explored the conditions for detergent solubilization of functionally active, heterologously expressed human TAP from microsomal membranes. The efficiency to solubilize TAP was tested for a variety of detergents as well as for different solubilization conditions. The activity of the solubilized TAP complex was analyzed over time, using a non‐radioactive crosslinking assay with a photo‐activateable peptide, in the presence or absence of external lipid. The detergent CHAPS was found optimally to retain activity and thus allowed us to reconstitute detergent‐solubilized, active TAP into proteoliposomes.
Insulin and cyclic AMP act at different levels on transcription of the L‐type pyruvate kinase gene
Authors: Cuif Marie-Hélène , Doiron Bruno , Kahn Axel
Published: July 2002

We previously demonstrated that, in hepatocytes in primary culture, the role of insulin on induction of L‐type pyruvate kinase (L‐PK) gene expression was mainly to induce glucokinase synthesis, needed for glucose phosphorylation to glucose 6‐phosphate. However, we show here that when hepatocytes have been isolated from rats starved for 72 h, glucose and constitutive glucokinase expression was not sufficient to fully stimulate the L‐PK promoter, low insulin concentrations being still required. In addition, activation remains sensitive to cAMP inhibition, but cannot be reproduced in the absence of insulin by a competitive cAMP antagonist. We propose that both insulin and cAMP act on expression of the L‐PK gene at, at least, two levels: positive and negative regulation of glucokinase gene expression, and more downstream levels.
Kinetic mechanism of active site non‐equivalence in transketolase
Authors: Kovina Marina V , Selivanov Vitaliy A , Kochevova Natalia V , Kochetov German A
Published: July 2002

The two‐step mechanism of coenzyme (TDP) binding to apotransketolase has been examined by kinetic modeling, and the rate and equilibrium constants for each binding step for two active sites have been determined. The dissociation constants for the primary fast binding step and the forward rate constants for the secondary slow binding step have been shown to be similar for two active sites. The backward rate constants for the secondary binding step are different for two active sites, providing the kinetic mechanism of their non‐equivalence in TDP binding.
Study of complexes of a tryptophan‐free mutant of E. coli trp aporepressor with tryptophan analogues using optically detected magnetic resonance (ODMR)
Authors: Ozarowski Andrzej , Wu Jie Qiang , Maki August H
Published: July 2002

Phosphorescence and optically detected magnetic resonance (ODMR) spectra of tryptophan (W) and several of its analogues (4‐, 5‐, 6‐methyltryptophan (MeW); 4‐, 5‐, 6‐fluorotryptophan (FW); 5‐bromotryptophan) are compared with those of complexes formed with the W‐free trp aporepressor from Escherichia coli (W19,99F). W19,99F binds W and each analogue except 4‐FW with an estimated K
D≤30 μM; triplet state spectroscopic and kinetic effects that accompany binding at the corepressor site are reported. ODMR data for the MeW isomers are presented for the first time. No binding of 7‐azaW is observed, in agreement with the low affinity found by previous workers.