bluuebunny/crossref_metadata_2025 huggingface dataset:
Nuclear and cytoplasmic α1(I) collagen mRNA‐binding proteins
Authors: Määttä Arto , Penttinen Risto P.K.
Published: July 2002

We have recently identified a cytoplasmic protein, α1‐RBF67, that specifically interacts with the conserved 3'‐untranslated region of the α1(I) collagen gene. The binding activity was decreased in extracts from dexamethasone treated cells, which correlates with the known accelerated turnover of the COL1A1 RNA [Määttä A. and Penttinen R.P.K. (1993) Biochem. J. 295, 691‐698]. Now we report that a very similar protein is present in nuclear extracts of NIH 3T3, human fibroblast and HeLa cells, which suggests that determination of cytoplasmic mRNA stability is not the sole function of the α1‐RBF67 activity. The binding to the RNA probe can be inhibited by annealing a DNA oligonucleotide or using excess of cold specific competitors. In UV‐cross linking assays the nuclear protein has the same molecular weight (67 kDa) as the cytoplasmic one and the RNA‐bound peptides generated by CNBr or V8 protease cleavage from both the cytoplasmic and the nuclear protein were identical. This protein was the only one of several nuclear collagen mRNA 3'‐UTR binding proteins that was present in both nuclear and cytoplasmic extracts. In fibroblasts heparin‐resistant nuclear RNA binding proteins had molecular weights of 45, 67 (α1‐RBF67), and 71 kDa. HeLa‐cells contained an additional protein of 51 kDa and several non‐specific RNA‐binding proteins. The binding activity is modified by changes in the redox state, which implicates that in the nucleus the binding affinities of α1(I) collagen RNA‐binding protein and AP‐1, a redox sensitive nuclear factor, that is important in the transcription of α1(I) collagen gene, can be regulated simultaneously to the same direction.
Cloning and sequence analysis of a cDNA encoding rice glutaredoxin
Authors: Minakuchi Kazunobu , Yabushita Tetsushige , Masumura Takehiro , Ichihara Ken'ichi , Tanaka Kunisuke
Published: July 2002

A full‐length cDNA clone (RASC8) encoding glutaredoxin (thioltransferase) was isolated from a cDNA library of an aleurone layer prepared from a developing seed of rice (Oryza sativa L.). RASC8, 568bp in length, contained an ATG codon and two possible polyadenylation signals, and encoded 112 amino acid residues. Cys‐Pro‐Phe‐Cys, which is the active site and a highly conserved sequence among thioltransferases, was found in the deduced amino acid sequence. RASC8 was introduced into an expression vector pMALc2 and the translated product possessed thioltransferase activity.
Tumour necrosis factor is in equilibrium with a trimeric molten globule at low pH
Authors: Hlodan Roman , H. Pain Roger
Published: July 2002

The reversible acid denaturation of tumour necrosis factor, TNFα, a trimeric, all‐β protein, leads to significant conformational changes within the molecule. A change in far UV CD spectra reveals a shift in the secondary structure content of the protein, with α‐helical structure being induced. Loss of ellipticity in the near UV reflects a loss of tertiary interactions. This form of TNF is both compact and trimeric, as revealed by fluorescence anisotropy and sedimentation velocity analysis, respectively. Acid‐denatured TNF therefore possesses the defining features of the molten globule intermediate while retaining the ability of the still incompletely folded monomers to exhibit the surface specificity necessary for maintaining the trimeric state.
Crel, the carbon catabolite repressor protein from Trichoderma reesei
Authors: Strauss Joseph , Mach Robert L. , Zeilinger Susanne , Hartler Gernot , Stöffler Georg , Wolschek Markus , Kubicek C.P.
Published: July 2002

In order to investigate the mechanism of carbon catabolite repression in the industrially important fungus Trichoderma reesei, degenerated PCR‐primers were designed to amplify a 0.7‐bp fragment of the crel gene, which was used to clone the entire gene. It encodes a 402‐amino acid protein with a calculated M
r, of 43.6 kDa. Its aa‐sequence shows 55.6% and 54.7% overall similarity to the corresponding genes of Aspergillus nidulans and A. niger, respectively. Similarity was restricted to the aa‐region containing the C2H2 zinc finger and several aa‐regions rich in proline and basic amino acids, which may be involved in the interaction with other proteins. Another aa‐region rich in the SPXX‐motif that has been considered analogous to a region of yeast RGR1p, was instead identified as a domain occurring in several eucaryotic transcription factors. The presence of the crel translation product was demonstrated with polyclonal antibodies against Cre1, which identified a protein of 43 (±2) kDa in cell‐free extracts from T. reesei. A Cre1 protein fragment from the two zinc fingers to the region similar to the aa‐sequence of eucaryotic transcription factors, was expressed in Escherichia coli as a fusion protein with glutathione S‐transferase. EMSA and in vitro footprinting revealed binding of the fusion protein to the sequence 5′‐GCGGAG‐3′, which matches well with the A. nidulans consensus sequence for CreA binding (5′‐SYGGRG‐3′). Cell‐free extracts of T. reesei formed different complexes with DNA‐fragments carrying this binding sites, and the presence of Cre1 and additional proteins in these complexes was demonstrated. We conclude that T. reesei Cre1 is the functional homologue of Aspergillus CreA and that it binds to its target sequence probably as a protein complex.
Triplex‐mediated cleavage of DNA by 1,10‐phenanthroline‐linked 2′‐O‐methyl RNA
Authors: Shimizu Miho , Morioka Hiroshi , Inoue Hideo , Ohtsuka Eiko
Published: July 2002

We have previously reported that 2′‐O‐methyl RNAs are efficient probes for duplex DNA. Here we describe the design, synthesis, and DNA cleaving activity of 1,10‐phenanthroline (OP)‐linked 2′‐O‐methyl RNA (OP‐m). Although a local triple helix was formed, both with OP‐m and a control OP‐linked DNA at the target sequence of the duplex DNA, the promoter region of the human thrombomodulin gene, the cleavage efficiencies on both strands were not proportional when OP‐m was used as a cleavage agent. These results may reflect the structural differences of the respective triple helices and the duplex‐triplex junction, formed from the two types of triplex‐forming oligonucleotides, 2′‐O‐methyl RNA and DNA. Since the OP‐ms were found to work as preferential purine‐strand cutters for duplex DNA, they would be useful as unique tools for genome analysis.
The C‐terminal domain of peptide deformylase is disordered and dispensable for activity
Authors: Meinnel Thierry , Lazennec Christine , Dardel Frederic , Schmitter Jean-Marie , Blanquet Sylvain
Published: July 2002

Upon trypsinolysis, the 18 C‐terminal residues of Escherichia coli peptide deformylase were removed but the resulting form exhibited full activity. Moreover, a mutant fms gene encoding the first 145 out of the 168 residues of the enzyme was able to complement a fms(Ts) strain and exhibited full activity. Upon progressive truncation up to residue 139, both activity and stability decreased up to complete inactivation. Mutagenesis of residues of the 138–145 region highlights the importance of Leu‐141 and Phe‐142. N‐Terminal deletions were also carried out. Beyond two residues off, the enzyme showed a dramatic instability. Finally, NMR and thermostability studies of the full‐length enzyme and comparison to the 1–147 form strongly suggest that the dispensable residues are disordered in solution.
The 32 kDa tonoplast polypeptide Di associated with the V0‐type H+‐ATPase of Mesembryanthemum crystallinum L. in the CAM state: A proteolytically processed subunit B?
Authors: Zhigang An , Löw Rainer , Rausch Thomas , Lüttge Ulrich , Ratajczak Rafael
Published: July 2002

In the facultative halophyte Mesembryanthemum crystallinum, the salt‐ or age‐induced transition to crassulacean acid metabolism (CAM) leads to the occurrence of a tonoplast‐bound 32 kDa polypeptide (Di). The alignment of its N‐terminal protein sequence with protein sequences of recently cloned higher plant V‐ATPase B‐subunits indicates that Di may be derived from subunit B by proteolytic removal of a protein fragment of about 20 kDa from its N‐terminus. Furthermore, an antiserum directed against Di cross‐reacts with subunit B from Nicotiana tabacum. It inhibits both proton pumping and ATP hydrolysis of the holoenzyme in M. crystallinum. As Di remains firmly attached to the holoenzyme the proteolytic processing may have functional implications.
Recombinant human proteinase 3, the Wegener's autoantigen, expressed in HMC‐1 cells is enzymatically active and recognized by c‐ANCA
Authors: Specks Ulrich , Fass David N. , Fautsch Michael P. , Hummel Amber M. , Viss Margaret A.
Published: July 2002

We developed a stable expression system for conformationally intact recombinant human PR3 (rPR3) using the human mast cell line HMC‐1. Like in U937 cells, the rPR3 is processed from a 34 kDa precursor to the 29 kDa mature form, primarily as the result of oligosaccharide trimming. The rPR3 binds [3H]DFP and hydrolyzes the substrate N‐methoxysuccinyl‐Ala‐Ala‐Ala‐Pro‐Val‐pNA. The enzymatic activity is inhibited by greater than 95% by α1‐PI. The rPR3 and the enzymatically inactive mutant rPR3‐S176A are both packaged in granules. Thus, proteolytic autoprocessing is not required for PR3's targeting to granules. This rPR3 is the first to be recognized by most c‐ANCA from WG patients and all anti‐PR3 ANCA that were detected by standard anti‐PR3 specific ELISA. This expression system for rPR3 represents a versatile tool for the analysis of its intracellular processing, structure‐function relationships and interaction with autoantibodies.
Screening for gestational diabetes mellitus in Korea
Authors: Jang H.C. , Cho N.H. , Jung K.B. , Oh K.S. , Dooley S.L. , Metzger B.E.
Published: July 2002

Objectives: To examine the effect of clinical characteristics on the prevalence of gestational diabetes mellitus (GDM) and to find the most effective screening program for GDM in Korea. Methods: Universal screening with a 50‐g glucose load at 24–28 weeks' gestation, as recommended by the Third International Workshop‐Conference on Gestational Diabetes Mellitus, was carried out among 3581 consecutive Korean women. Women with a 1‐h plasma glucose ≥ 130 mg/dl underwent a 3‐h 100‐g oral glucose tolerance test. The women's clinical characteristics and risk factors for GDM were recorded at the time of the screening test. Results: The overall prevalence of GDM in Korean women was 2.2 cases/100. Although only 1.3% of this population was obese, the prevalence of GDM was found to be significantly increased with increasing body mass index. When 135 mg/dl is used as a threshold, the number of women requiring a diagnostic test decreases to 19.5%, enabling identification of 98.8% of women with GDM. Conclusion: Universal screening using 135 mg/dl as a threshold and early screening of those with two or more risk factors represent the most effective paradigm for Korea.
Is the manganese stabilizing 33 kDa protein of photosystem II attaining a ‘natively unfolded’ or ‘molten globule’ structure in solution?
Authors: Shutova T. , Irrgang K.-D. , Klimov V.V. , Renger G.
Published: July 2002

This study compares the properties of the extrinsic 33 kDa subunit acting as ‘manganese stabilizing protein’ (MSP) of the water oxidizing complex with characteristic features of proteins that are known to attain a ‘natively unfolded’ or a ‘molten globule’ structure. The analysis leads to the conclusion that the MSP in solution is most likely a ‘molten globule’ with well defined compact regions of β structure. The possible role of these structural peculiarities of MSP in solution for its function as important constituent of the WOC is discussed.
Rap1‐suppressed tumorigenesis is concomitant with the interference in Ras effector signaling
Authors: Lin Yea-Lih , Mettling Clément , Chou Chen-Kung
Published: July 2002

Expression of Rap1 blocks epithelial growth factor‐induced extracellular signal‐regulated kinases (ERKs) activation. However, recent studies demonstrated that Rap1 mediates ERKs activation induced by nerve growth factor. The anti‐oncogenic effect of Rap1 has been reported but its mechanism remains unclear. To evaluate the correlation between the anti‐transforming effect and the activation of ERKs, we transfected rap1 cDNA into Hep3B cells and selected stable transfectants. The Rap1 transfectants completely lost their intrinsic tumorigenicity in Balb/c nude mice. Both insulin and 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA)‐stimulated ERK activations were also blocked. Our findings suggest that Rap1‐suppressed tumorigenicity is concomitant with ERKs inhibition.
Structural domains of the insulin receptor and IGF receptor required for dimerisation and ligand binding
Authors: Molina L , Marino-Buslje C , Quinn D.R , Siddle K
Published: July 2002

We investigated structural requirements for dimerisation and ligand binding of insulin/IGF receptors. Soluble receptor fragments consisting of N‐terminal domains (L1/CYS/L2, L1/CYS/L2/F0) or fibronectin domains (F0/F1/F2, F1/F2) were expressed in CHO cells. Fragments containing F0 or F1 domains were secreted as disulphide‐linked dimers, and those consisting of L1/CYS/L2 domains as monomers. None of these proteins bound ligand. However, when a peptide of 16 amino acids from the α‐subunit C‐terminus was fused to the C‐terminus of L1/CYS/L2, the monomeric insulin and IGF receptor constructs bound their respective ligands with affinity only 10‐fold lower than native receptors.
A synapomorphic disulfide bond is critical for the conformational stability and cytotoxicity of an amphibian ribonuclease
Authors: Leland Peter A. , Staniszewski Kristine E. , Kim Byung-Moon , Raines Ronald T.
Published: July 2002

Onconase® (ONC) is a homolog of ribonuclease A (RNase A) that has unusually high conformational stability and is toxic to human cancer cells in vitro and in vivo. ONC and its amphibian homologs have a C‐terminal disulfide bond, which is absent in RNase A. Replacing this cystine with a pair of alanine residues greatly decreases the conformational stability of ONC. In addition, the C87A/C104A variant is 10‐fold less toxic to human leukemia cells. These data indicate that the synapomorphic disulfide bond of ONC is an important determinant of its cytotoxicity.
A 45 kDa protein related to PPARγ2, induced by peroxisome proliferators, is located in the mitochondrial matrix
Authors: Casas François , Domenjoud Lionel , Rochard Pierrick , Hatier Renée , Rodier Anne , Daury Laetitia , Bianchi Arnaud , Kremarik-Bouillaud Pascaline , Becuwe Philippe , Keller Jean-Marie , Schohn Hervé , Wrutniak-Cabello Chantal , Cabello Gérard , Dauça Michel
Published: July 2002

Besides their involvement in the control of nuclear gene expression by activating several peroxisome proliferator‐activated receptors (PPARs), peroxisome proliferators influence mitochondrial activity. By analogy with the previous characterization of a mitochondrial T3 receptor (p43), we searched for the presence of a peroxisome proliferator target in the organelle. Using several antisera raised against different domains of PPARs, we demonstrated by Western blotting, immunoprecipitation and electron microscopy experiments, that a 45 kDa protein related to PPARγ2 (mt‐PPAR) is located in the matrix of rat liver mitochondria. In addition, we found that the amounts of mt‐PPAR are increased by clofibrate treatment. Moreover, in EMSA experiments mt‐PPAR bound to a DR2 sequence located in the mitochondrial D‐loop, by forming a complex with p43. Last, studies of tissue‐specific expression indicated that mt‐PPAR is detected in mitochondria of all tissues tested except the brain in amounts positively related to p43 abundance.
The phytotoxic lichen metabolite, usnic acid, is a potent inhibitor of plant p‐hydroxyphenylpyruvate dioxygenase
Authors: Romagni Joanne G. , Meazza Giovanni , Nanayakkara N.P.Dhammika , Dayan Franck E.
Published: July 2002

The lichen secondary metabolite usnic acid exists as a (−) and a (+) enantiomer, indicating a α or β projection of the methyl group at position 9b, respectively. (−)‐Usnic caused a dose‐dependent bleaching of the cotyledonary tissues associated with a decrease of both chlorophylls and carotenoids in treated plants whereas no bleaching was observed with the (+) enantiomer. (−)‐Usnic acid inhibited protophorphyrinogen oxidase activity (I
50=3 μM), but did not lead to protoporphyrin IX accumulation. Bleaching appears to be caused by irreversible inhibition of the enzyme 4‐hydroxyphenylpyruvate dioxygenase by (−)‐usnic acid (apparent IC50=50 nM).
Synthetically prepared Amadori‐glycated phosphatidylethanolamine can trigger lipid peroxidation via free radical reactions
Authors: Oak Jeong-Ho , Nakagawa Kiyotaka , Miyazawa Teruo
Published: July 2002

This study for the first time confirmed the peroxidative role of the Amadori product derived from the glycation of phosphatidylethanolamine (PE), namely Amadori–PE. The product was synthesized from the reaction of dioleoyl PE with D‐glucose, and then purified by a solid‐phase extraction procedure, which was a key step in the next HPLC technique for the isolation of essentially pure Amadori–PE. When the synthetically prepared Amadori–PE was incubated with linoleic acid in the presence of Fe3+ in micellar system, a remarkable formation of thiobarbituric acid reactive substances was observed together with increases in lipid hydroperoxides. In addition, the lipid peroxidation caused by Amadori–PE was effectively inhibited by superoxide dismutase, mannitol, catalase and metal chelator. These results indicated that Amadori–PE triggers oxidative modification of lipids via the generation of superoxide, and implied the involvement of ‘lipid glycation’ along with membrane lipid peroxidation in the pathogenesis of diabetes and aging.
Functional identification of the glycerol permease activity of Arabidopsis thaliana NLM1 and NLM2 proteins by heterologous expression in Saccharomyces cerevisiae
Authors: Weig Alfons R. , Jakob Christiane
Published: July 2002

NLM proteins (NOD26‐like major intrinsic proteins) from plants contain amino acid sequence signatures which can be found in aquaporins including plant plasma membrane intrinsic proteins and tonoplast intrinsic proteins and glycerol permeases such as the Escherichia coli GlpF and the yeast FPS1 proteins. Heterologous expression of two members of the NLM subgroup from Arabidopsis thaliana (AtNLM1 and AtNLM2) in baker's yeast demonstrated the glycerol permease activity in addition to the previously described aquaporin activity of AtNLM1. The transport was non‐saturable up to 100 mM extracellular glycerol concentration. Longer‐chain sugar alcohols did not compete with the transport of radiolabelled glycerol and hexoses were also not transported through the pore.
Structure/function of human herpesvirus‐8 MIP‐II (1–71) and the antagonist N‐terminal segment (1–10)
Authors: Crump Matthew P , Elisseeva Elena , Gong Jiang-Hong , Clark-Lewis Ian , Sykes Brian D
Published: July 2002

Kaposi's sarcoma‐associated herpesvirus encodes a chemokine called vMIP‐II that has been shown to be a broad range human chemokine receptor antagonist. Two N‐terminal peptides, vMIP‐II(1–10) and vMIP‐II(1–11)dimer (dimerised through Cys11) were synthesised. Both peptides are shown to bind the CXC chemokine receptor 4 (CXCR4). vMIP‐II(1–10) was 1400‐fold less potent than the native protein whilst the vMIP‐II(1–11)dimer was only 180‐fold less potent. In addition, both peptides are CXCR4 antagonists. Through analysis of non‐standard, long mixing time two‐dimensional nuclear Overhauser enhancement spectroscopy experiments, 13C relaxation data and amide chemical shift temperature gradients for the N‐terminus of vMIP‐II, we show that this region populates a turn‐like structure over residues 5–8, both in the presence and absence of the full protein scaffold. This major conformation is likely to be in fast exchange with other conformational states but it has not previously been detected in monomeric chemokine structures. This and other studies [Elisseeva et al. (2000) J. Biol. Chem. 275, 26799–26805] suggest that there may be a link between the structuring of the short N‐terminal chemokine peptides and their ability to bind their receptor.
CCK2 (CCKB/gastrin) receptor mediates rapid protein kinase D (PKD) activation through a protein kinase C‐dependent pathway
Authors: Chiu Terence , Rozengurt Enrique
Published: July 2002

Addition of gastrin or cholecystokinin octapeptide (CCK‐8) to cultures of Rat‐1 cells stably transfected with the CCK2 (CCKB/gastrin) receptor induced protein kinase D (PKD) activation that was detectable within 1 min and reached a maximum (∼10‐fold) after 2.5 min of hormonal stimulation. Half‐maximal PKD activation for both CCK‐8 and gastrin was achieved at 10 nM. Treatment with various concentrations of the selective PKC inhibitors Ro 31‐8220 or GF‐I potently blocked PKD activation induced by subsequent addition of CCK‐8 in a concentration‐dependent fashion. Our results indicate that PKC‐dependent PKD activation is a novel early event in the action of gastrin and CCK‐8 via CCK2 receptors.
Site‐directed mutagenesis evidence for a negatively charged trypsin inhibitory loop in sweet potato sporamin
Authors: Yao Pei-Li , Hwang Ming-Jing , Chen Yih-Ming , Yeh Kai-Wun
Published: July 2002

Sporamin, a sweet potato tuberous storage protein, has trypsin inhibitory activity. Sequence comparison with other plant trypsin inhibitors (TIs) of the Kunitz family reveals that, instead of the conserved Arg or Lys found in other Kunitz TIs, sporamin contains a negatively charged residue (Asp70 or Glu72) at the P1 reactive site. Using site‐directed mutagenesis, six mutants were generated containing substitutions at the reactive site and at one of the disulfide bonds, and the recombinant proteins were assayed for TI activity. Mutants Asp70Val and Glu72Arg were found to have only 2–3% of the wild‐type activity. These results provide the first evidence for a negatively charged trypsin inhibitory loop and a new mechanism of trypsin inhibition in the Kunitz family.
Coordination between transcription and pre‐mRNA processing
Authors: Cramer P. , Srebrow A. , Kadener S. , Werbajh S. , de la Mata M. , Melen G. , Nogués G. , Kornblihtt A.R.
Published: July 2002

A large body of work has proved that transcription by RNA polymerase II and pre‐mRNA processing are coordinated events within the cell nucleus. Capping, splicing and polyadenylation occur while transcription proceeds, suggesting that RNA polymerase II plays a role in the regulation of these events. The presence and degree of phosphorylation of the carboxy‐terminal domain of RNA polymerase II large subunit is important for functioning of the capping enzymes, the assembly of spliceosomes and the binding of the cleavage/polyadenylation complex. Nuclear architecture and gene promoter structure have also been shown to play key roles in coupling between transcription and splicing.
Molecular cloning of Drosophila Rh6 rhodopsin: the visual pigment of a subset of R8 photoreceptor cells1
Authors: Huber Armin , Schulz Simone , Bentrop Joachim , Groell Christine , Wolfrum Uwe , Paulsen Reinhard
Published: July 2002

By screening retinal cDNA libraries for photoreceptor‐specifically expressed genes we have isolated and sequenced a cDNA clone encoding the rhodopsin (Rh6) of a subset of R8 photoreceptor cells of the Drosophila compound eye. Compared to the other visual pigments of Drosophila, this rhodopsin is equally homologous to Rh1 and Rh2 (51% amino acid identity) but shows only 32% and 33% amino acid identity with Rh3 and Rh4, respectively. The open reading frame codes for a protein of 369 amino acids (MW=41 691). The primary structure of Rh6 displays sites typical for rhodopsin molecules in general, for example, a chromophore binding site in transmembrane domain VII, sequence motifs in the intracellular loops 2 and 3 required for the binding of a heterotrimeric G‐protein, and a glycosylation site near the N‐terminus which seems to be important for protein transport and maturation. Since R8 cells are founder cells in the developing compound eye, the isolation of a rhodopsin gene expressed in these cells may aid the understanding of terminal differentiation of photoreceptor cells.
Comparative processing of bovine leukemia virus envelope glycoprotein gp72 by subtilisin/kexin‐like mammalian convertases
Authors: Zarkik Sarra , Decroly Etienne , Wattiez Ruddy , Seidah Nabil G , Burny Arsène , Ruysschaert Jean-Marie
Published: July 2002

Intracellular proteolytic processing of bovine leukemia virus (BLV) envelope glycoprotein precursor (gp72) at the C‐terminal end of the RVRR268↓ site is an essential step for virus infectivity. Subtilisin/kexin‐like convertases cleave proproteins at preferred RX(K/R)R↓ sites, including those commonly found in viral envelope glycoprotein precursors. We first demonstrated that gp72 is processed into gp51/gp30 in both CV1 cells and the furin‐deficient LoVo cells, leading us to compare the ability of mammalian convertases to cleave BLV gp72 in vitro. In contrast to the inability of the neuroendocrine PC1 to cleave gp72, the convertases furin, PACE4, PC5‐A and PC5‐B, which process constitutively secreted precursors, can effectively cleave gp72 into gp51/gp30. N‐terminal sequence analysis of the convertase‐generated gp30 demonstrated that cleavage occurs at the in vivo‐utilized RVRR↓SPV site. Such furin‐, PACE4‐ and PC5‐mediated processing was completely inhibited by the α1‐antitrypsin variant α1‐PDX. Mutagenesis of the gp72 cleavage site into RVR
‐
PV resulted in complete abrogation of gp72 processing by endogenous CV‐1 cells and by convertases in vitro. Since our in vitro data suggest a redundancy in the ability of the convertases to cleave gp72, RT‐PCR analysis was used to define the convertases expressed in B‐lymphocytes, representing one of the major targets of BLV infection. Our data revealed that only furin and the newly discovered PC7 mRNAs are expressed in Raji, B‐Jab and LG2 cell lines.
The effect of neuropeptides kyotorphin and neokyotorphin on proliferation of cultured brown preadipocytes
Authors: Bronnikov Gennady , Dolgacheva Ludmila , Zhang Shi-Jin , Galitovskaya Elena , Kramarova Ludmila , Zinchenko Valery
Published: July 2002

Stimulation of DNA and protein synthesis in brown preadipocytes by 1 μM neokyotorphin in serum‐containing media was comparable with the effect of 1 μM norepinephrine. In serum‐free medium a decrease and a shift of the maximal effect to lower concentration of neokyotorphin were observed. Kyotorphin had no effect on cell proliferation in either medium; however, 0.01–1 μM kyotorphin inhibited the cell proliferation stimulated by 1 μM norepinephrine. Norepinephrine and both peptides stimulated comparable Ca2+ rise in freshly isolated brown preadipocytes. The effects of neokyotorphin and norepinephrine were additive, whereas 0.03–0.3 μM kyotorphin blocked the action of 3 μM norepinephrine. The peptides did not affect the cAMP level in non‐stimulated or norepinephrine‐stimulated cultured cells. The effects of the peptides on the brown fat cell cultures indicate that peripheral tissue cells contain receptors for these neuropeptides.
The T transcription factor functions as a dimer and exhibits a common human polymorphism Gly‐177‐Asp in the conserved DNA‐binding domain
Authors: Papapetrou C. , Edwards Y.H. , Sowden J.C.
Published: July 2002

T is a transcription factor which activates transcription by binding to repeated arrangements of the dodecamer 5′‐AGGTGTGAAATT‐3′. Using in vitro synthesised T protein, we have demonstrated that T binds to its target DNA as a homodimer and that truncated protein containing only the N‐terminal 233 amino‐acid residues, which comprise the DNA‐binding domain, can form a dimer. We also report a common human polymorphism, Gly‐177‐Asp, within the DNA‐binding domain at a position which is a conserved glycine residue in T homologues from other vertebrates. The proposition that T forms heterodimers with other members of the T‐box transcription factor family and the implications for disorders of axial development are discussed.
Intermittent divergence of the protein tyrosine kinase family during animal evolution
Authors: Suga Hiroshi , Kuma Kei-ichi , Iwabe Naoyuki , Nikoh Naruo , Ono Kanako , Koyanagi Mitsumasa , Hoshiyama Daisuke , Miyata Takashi
Published: July 2002

The protein tyrosine kinases (PTKs) are a large protein family consisting of many subfamilies with a variety of domain structures. The basic functions are thought to differ for different subfamilies. To know the dates at which the subfamilies diverged by gene duplications, a phylogenetic tree of the PTKs was inferred by comparing sequences from a wide range of species covering diploblasts and triploblasts. The PTK tree revealed that almost all of the gene duplications that gave rise to different subfamilies occurred rapidly before the diploblast–triploblast split, accompanying with rapid amino acid substitutions. This type of gene duplication was, however, rarely observed after that split. Long after the subfamily divergence, another type of gene duplication that gave rise to diverse tissue‐specific genes occurred in each subfamily on the chordate lineage since the separation from arthropods. This type of gene duplication occurred frequently before the fish–tetrapod split, accompanying with rapid amino acid substitutions. In contrast, both the frequency of gene duplications and the rate of the amino acid substitutions were considerably reduced after that split. These results strongly suggest that the PTKs diverged intermittently, but not gradually, during animal evolution.
Transient‐state and steady‐state kinetics of the oxidation of aliphatic and aromatic thiols by horseradish peroxidase
Authors: Burner U. , Obinger C.
Published: July 2002

In the course of oxidation of thiols by peroxidases thiyl radicals are formed which are known to undergo several free‐radical conjugative reactions, among others leading to hydrogen peroxide formation. The present paper for the first time presents a comparative transient‐state and steady‐state investigation of the reaction of 15 aliphatic and aromatic mono‐ and dithiols with horseradish peroxidase (HRP). Both sequential‐stopped‐flow spectrophotometric investigations of the reaction of HRP intermediates Compound I (k
2) and Compound II (k
3) with thiols and measurements of the overall thiol oxidation and the simultaneous oxygen consumption in the presence and absence of exogenously added hydrogen peroxide (10 μM) have been performed. With HRP as thiyl radical generator it was shown that three groups of thiols have to be distinguished: (i) Aromatic thiols (e.g. thiophenol, 2‐mercaptopurine) were excellent electron donors of both Compounds (k
2: 104–107 M−1 s−1 and k
3: 103–106 M−1 s−1); however, the overall reaction was shown to depend on addition of hydrogen peroxide, indicating insufficient peroxide regeneration by arylthiyl radicals. (ii) Aliphatic thiols which were extremely bad substrates (k
3 <10 M−1 s−1) for HRP (e.g. homocysteine, glutathione) and/or have a pK
a,SH>9.5 (e.g. N‐acetylcysteine, α‐lipoic acid) were also shown to depend on exogenously added H2O2 to maintain the peroxidasic reaction, whereas (iii) with those thiols with rates of k
3 between 11 and 1600 M−1 s−1 (e.g. cysteine, cysteamine, cysteine methyl ester, cysteine ethyl ester) and/or with a pK
a,SH <8 (penicillamine) thiol oxidation was independent of exogenously added hydrogen peroxide, indicating sufficient hydrogen peroxide regeneration.
Identification of a 48‐kDa prenylated protein that associates with microtubules as 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase in FRTL‐5 cells
Authors: Laezza Chiara , Wolff J , Bifulco Maurizio
Published: July 2002

In an effort to study the nature of tubulin attachment to membranes, we have previously observed that after blocking prenylation in FRTL‐5 thyroid cells, the microtubules become disconnected from the plasma membrane region [Bifulco M. et al. (1983) J. Cell. Physiol. 155, 340–348]. In this study we show that several [3H]mevalonate labeled proteins in FRTL‐5 cells associate with membrane and cytoskeleton and, among these, we describe the presence of a 48‐kDa prenylated protein, identified by immunoprecipitation as 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP), that associates with microtubules. This latter association persists through several polymerization/depolymerization cycles, whereas other prenylated proteins are lost. It is suggested that CNP can be a novel microtubule‐associated protein (MAP) and a promising candidate as a membrane anchor for microtubules.
Glyoxalase II from A. thaliana requires Zn(II) for catalytic activity
Authors: Crowder Michael W. , Maiti Mrinal K. , Banovic Linda , Makaroff Christopher A.
Published: July 2002

Cytosolic glyoxalase II from Arabidopsis thaliana, GLX2‐2, was overexpressed and purified to homogeneity using Q‐sepharose chromatography. MALDI‐TOF mass spectrometry studies indicated a molecular weight of 28 767 Da. Using steady‐state kinetics studies, the purified enzyme exhibited a K
m of 660±100 μM and a k
cat of 484±92 s−1 at 37°C. Metal analyses demonstrated that the enzyme binds 2.1±0.5 moles of Zn(II) per monomer; the binding of Zn(II) is essential for enzyme viability and activity. Sequence comparison of glyoxalase II enzymes from human, A. thaliana, and yeast and the metallo‐β‐lactamases reveal that all metal binding ligands of the metallo‐β‐lactamases are conserved in glyoxalase II enzymes, suggesting that all glyoxalase II enzymes are Zn(II) metalloenzymes. These results and their implications are discussed in light of previous studies on glyoxalase II, and an active site for the glyoxalase II enzymes is proposed.
Transport of glutathione prostaglandin A conjugates by the multidrug resistance protein 1
Authors: Evers Raymond , Cnubben Nicole H.P , Wijnholds Jan , van Deemter Liesbeth , van Bladeren Peter J , Borst Piet
Published: July 2002

The human multidrug resistance protein MRP1 mediates transport of organic substrates conjugated to glutathione, glucuronide, or sulfate. The naturally occurring prostaglandins A1 and A2 can form two diastereomeric glutathione S‐conjugates, and it has been speculated that these might be substrates for MRP1. Here we present evidence that polarized MDCKII cells expressing MRP1 cDNA transport PGA1‐GS to the basolateral side of a cell monolayer, in accordance with the lateral localization of human MRP1 in these cells. Furthermore, we show that vesicles made from yeast cells expressing MRP1 cDNA and from mouse erythrocytes (known to contain mrp1) actively accumulate both diastereomers of PGA2‐GS with a similar efficiency. Recently, we generated mice with a homozygous mutant mrp1 allele. Uptake of PGA2‐GS in vesicles made from erythrocytes of these mice was 3.2 times lower than in wild‐type vesicles, but was still significantly above background. This residual transport activity was partly inhibited by methotrexate and cAMP, whereas mrp1‐mediated activity was unaffected by these compounds. We conclude that mouse erythrocytes contain at least two transport systems for PGA2‐GS. One of these is mrp1; the other one has not been identified yet, but can be inhibited by methotrexate and cAMP.
Aggregation of β‐lactoglobulin and influence of D2O
Authors: Verheul Marleen , Roefs Sebastianus P.F.M. , de Kruif Kees G.
Published: July 2002

The conformational stability of β‐lactoglobulin increases in D2O over that in H2O. This is concluded from an increase in peak temperature by about 3°C of differential scanning calorimetry (DSC) thermograms and from a decrease in overall aggregation rate. However, effects of pH and salt concentration on the heat‐induced aggregation (reaction kinetics, DSC thermograms and aggregate growth) are similar in H2O and D2O. This indicates that the mechanism of heat‐induced aggregation of β‐lactoglobulin is not significantly affected by replacement of H2O with D2O.
Thylakoid protein phosphorylation in evolutionally divergent species with oxygenic photosynthesis
Authors: Pursiheimo Saijaliisa , Rintamäki Eevi , Baena-Gonzalez Elena , Aro Eva-Mari
Published: July 2002

Phosphothreonine antibody was used to explore reversible thylakoid protein phosphorylation in vivo in evolutionally divergent organisms with oxygenic photosynthesis. Three distinct groups of organisms were found. Cyanobacteria and red algae, both with phycobilisome antenna system, did not show phosphorylation of any of the photosystem II (PSII) proteins and belong to group 1. Group 2 species, consisting of a moss, a liverwort and a fern, phosphorylated both the light‐harvesting chlorophyll a/b proteins (LHCII) and the PSII core proteins D2 and CP43, but not the D1 protein. Reversible phosphorylation of the D1 protein seems to be the latest event in the evolution of PSII protein phosphorylation and was found only in seed plants, in group 3 species. Light‐intensity‐dependent regulation of LHCII protein phosphorylation was similar in group 2 and 3 species, with maximal phosphorylation of LHCII at low light and nearly complete dephosphorylation at high light.
Translocation of β crystallin in neural cells in response to stress
Authors: Coop Audrey , Wiesmann Kirsten E.H , Crabbe M.James C
Published: July 2002

While extralenticular expression of proteins in the α crystallin (small heat shock protein) family is well documented, that for proteins in the β/γ‐superfamily is less well established. Here we show, using SDS‐PAGE, Western blotting and confocal microscopy, that there is a constitutive level of β crystallin expression in mouse N1E‐115 neural cells. Furthermore, upon heat shock at 43°C or 55°C, or cold shock at 30°C, β crystallin immunoreactivity translocated predominantly from the nuclear region into the cytoplasmic region of the cells. In conditions of stress, it may be important for β crystallin to be recruited into the cytoplasm to stabilise other proteins via its high β‐sheet content, and/or to ensure that storage levels of cytoplasmic Ca2+ are maintained.
Cloning and expression of a new cDNA from monocotyledonous plants coding for a diadenosine 5′,5′′′‐P1,P4‐tetraphosphate hydrolase from barley (Hordeum vulgare)
Authors: Churin Juri , Hause Bettina , Feussner Ivo , Maucher Helmut P , Feussner Kirstin , Börner Thomas , Wasternack Claus
Published: July 2002

From a cDNA library generated from mRNA of white leaf tissues of the ribosome‐deficient mutant ‘albostrians' of barley (Hordeum vulgarecv. Haisa) a cDNA was isolated carrying 54.2% identity to a recently published cDNA which codes for the diadenosine‐5′,5′′′‐P1,P4‐tetraphosphate (Ap4A) hydrolase ofLupinus angustifolius(Maksel et al. (1998) Biochem. J. 329, 313–319), and 69% identity to four partial peptide sequences of Ap4A hydrolase of tomato. Overexpression inEscherichia colirevealed a protein of about 19 kDa, which exhibited Ap4A hydrolase activity and cross‐reactivity with an antibody raised against a purified tomato Ap4A hydrolase (Feussner et al. (1996) Z. Naturforsch. 51c, 477–486). Expression studies showed an mRNA accumulation in all organs of a barley seedling. Possible functions of Ap4A hydrolase in plants will be discussed.
Monomeric bovine β‐lactoglobulin adopts a β‐barrel fold at pH 2
Authors: Fogolari F , Ragona L , Zetta L , Romagnoli S , De Kruif K.G , Molinari H
Published: July 2002

We have determined a crude structure of the apo form of bovine β‐lactoglobulin, a protein of 162 amino acid residues with a molecular mass of 18 kDa, at a low pH on the basis of data collected using only homonuclear 1H NMR spectroscopy. An ensemble of protein conformations was calculated with the distance‐geometry algorithm for NMR applications (DYANA). The monomeric protein at low pH adopts a β‐barrel fold, well‐superimposable on the structure determined by X‐ray crystallography for the dimer at physiological pH. NMR evidence suggests the presence of disordered loop regions and terminal segments. Structural differences between the monomer at pH 2 and the dimer at pH 7, obtained by X‐ray crystallography, are discussed, paying particular attention to surface electrostatic properties, in view of the high charge state of the protein at low pH.
p53 and the ribosomal protein L5 participate in high molecular mass complex formation with protein kinase CK2 in murine teratocarcinoma cell line F9 after serum stimulation and cisplatin treatment
Authors: Guerra Barbara , Issinger Olaf-Georg
Published: July 2002

Using the murine teratocarcinoma cell line F9 we investigated the influence of serum stimulation and cisplatin treatment on the p53, CK2, MDM2 levels. Both treatments led to an increase of p53, though with different kinetics; the other proteins investigated were not affected. We present direct evidence by immunoprecipitation for an association of protein kinase CK2 holoenzyme (α2β2), p53, and the ribosomal protein L5. The results suggest complexes between the CK2 holoenzyme and p53 but also p53/CKβ complexes. Furthermore we provide evidence for the existence of high molecular mass complexes of CK2 in vivo. This is the first evidence that, under physiological conditions, protein kinase CK2 does not exist solely as a heterotetramer, but predominantly in association with other proteins.
Structure and mutation analysis of the glycogen storage disease type 1b gene
Authors: Marcolongo Paola , Barone Virginia , Priori Giuseppina , Pirola Barbara , Giglio Sabrina , Biasucci Giacomo , Zammarchi Enrico , Parenti Giancarlo , Burchell Ann , Benedetti Angelo , Sorrentino Vincenzo
Published: July 2002

Glycogen storage disease (GSD) 1b is the deficiency of endoplasmic reticulum glucose‐6‐phosphate (G6P) transport. We here report the structure of the gene encoding a protein likely to be responsible for G6P transport, and its mapping to human chromosome 11q23.3. The gene is composed of nine exons spanning a genomic region of approximately 4 kb. Primers based on the genomic sequence were used in single strand conformation polymorphism (SSCP) analysis and mutations were found in six out of seven GSD 1b patients analysed.
Cloning of murine low molecular weight phosphotyrosine protein phosphatase cDNA: identification of a new isoform
Authors: Magherini F , Giannoni E , Raugei G , Cirri P , Paoli P , Modesti A , Camici G , Ramponi G
Published: July 2002

The low molecular weight phosphotyrosine protein phosphatase (LMW‐PTP) is a 18 kDa cytosolic enzyme, involved in the negative regulation of cell proliferation. In different mammalian species LMW‐PTPs are expressed in two molecular forms produced from a single primary transcript through an alternative splicing mechanism. In this paper we report the cloning, expression and characterization of mouse isoforms of LMW‐PTPs (called m‐IF1 and m‐IF2), very similar to the corresponding rat and human isoenzymes. Moreover we have identified a third cDNA encoding a protein (m‐IF2P) that presents three substitutions compared to m‐IF2. This new isoform is still active on pNPP, although to a lower extent: this reduction is mainly due to the leucine to proline substitution in position 13, within the catalytic loop. The mRNA expression level of this isoform is comparable to those of m‐IF1 and m‐IF2. It is likely that a gene duplication process followed by mutations has generated this new gene.
Creatine supplementation improves intracellular Ca2+ handling and survival in mdx skeletal muscle cells
Authors: Pulido S.M , Passaquin A.C , Leijendekker W.J , Challet C , Wallimann T , Rüegg U.T
Published: July 2002

Dystrophic skeletal muscle cells from Duchenne muscular dystrophy (DMD) patients and mdx mice exhibit elevated cytosolic Ca2+ concentrations ([Ca2+]c). Pretreatment of mdx myotubes for 6–12 days with creatine (20 mM) decreased the elevation in [Ca2+]c induced by either high extracellular Ca2+ concentrations or hypo‐osmotic stress to control levels. 45Ca2+ influx measurements suggest that creatine lowered [Ca2+]c by stimulating sarcoplasmic reticulum Ca2+‐ATPase. Creatine pretreatment increased levels of phosphocreatine but not ATP. Furthermore, myotube formation and survival were significantly enhanced by creatine pretreatment. Therefore, creatine supplementation may be useful for treatment of DMD.
AtMSI4 and RbAp48 WD‐40 repeat proteins bind metal ions1
Authors: Kenzior Alexander L , Folk William R
Published: July 2002

The mammalian RbAp48 protein is the most extensively studied member of the conserved family of Msi1‐like WD‐40 repeat proteins, which are components of complexes involved in the assembly and modification of chromatin. We have isolated a plant homolog of RbAp48, AtMSI4. By metal affinity chromatography, zinc blotting and atomic absorption analysis, we demonstrate that purified recombinant RbAp48 and AtMSI4 proteins bind 3–4 metal ions per molecule of protein. Metal competition assays indicate a preference for zinc. Both N‐ and C‐terminal halves of RbAp48 and AtMSI4 display zinc binding activity, suggesting it is an intrinsic property of the propeller structures likely to be formed by these proteins. Metal binding might mediate and/or regulate protein‐protein interactions which are functionally important in chromatin metabolism.
A third fibronectin type III domain in the extracellular region of the insulin receptor family
Authors: Marino-Buslje Cristina , Mizuguchi Kenji , Siddle Kenneth , Blundell Tom L.
Published: July 2002

The insulin receptor family consists of the homologous tyrosine kinase receptors, insulin receptor (IR), insulin‐like growth factor 1 receptor (IGF1R) and insulin receptor‐related receptor. The three‐dimensional structures of the tyrosine kinase domain of the IR and the first three extracellular domains (L1, Cys‐rich and L2) of the IGF1R are known. Here we present evidence that the connecting domain of the IR family is a member of the fibronectin type III (FnIII) superfamily. Structure‐based alignment of FnIII domains reveals several key residues that are also conserved in the sequence of the connecting domain. The alignment of the connecting domain with FnIII domains is in good agreement with secondary structure prediction. A model of the connecting domain shows a hydrophobic core formed by the conserved residues and is consistent with previously known biochemical data. This suggests that the IR family contains three FnIII domains in tandem in the extracellular juxtamembrane region.
Expression of Mash1 in basal cells of rat circumvallate taste buds is dependent upon gustatory innervation
Authors: Seta Yuji , Toyono Takashi , Takeda Shinobu , Toyoshima Kuniaki
Published: July 2002

Mash1, a mammalian homologue of the Drosophila achaete‐scute proneural gene complex, plays an essential role in differentiation of subsets of peripheral neurons. In this study, using RT‐PCR and in situ RT‐PCR, we investigated if Mash1 gene expression occurs in rat taste buds. Further, we examined dynamics of Mash1 expression in the process of degeneration and regeneration in denervated rat taste buds. In rat tongue epithelium, Mash1 gene expression is confined to circumvallate, foliate, and fungiform papilla epithelia that include taste buds. In taste buds, Mash1‐expressing cells are round cells in the basal compartment. In contrast, the mature taste bud cells do not express the Mash1 gene. Denervation and regeneration experiments show that the expression of Mash1 requires gustatory innervation. We conclude that Mash1 is expressed in cells of the taste bud lineage, and that the expression of Mash1 in rat taste buds is dependent upon gustatory innervation.