bluuebunny/crossref_metadata_2025 huggingface dataset:
Two‐dimensional 1H‐NMR study of bacterioopsin‐(34–65)‐polypeptide conformation
Authors: Arseniev Alexander S. , Maslennikov Innokenti V. , Bystrov Vladimir F. , Kozhich Alexander T. , Ivanov Vadim T. , Ovchinnikov Yuri A.
Published: July 2002

Conformation of the synthetic 32‐residue polypeptide, an analog of membrane spanning segment B (residues 34–65) of the Halobacterium halobium bacteriorhodopsin in the membrane mimetic system, methanol/chloroform (1:1), was investigated by 1H‐NMR spectroscopy. Previously it was shown by 19F‐NMR spectroscopy that this medium retains the native conformation of membrane bound BR and its fragments. The spectrum resonance was assigned by means of the sequential signal assignment porcedure using phase‐sensitive DQF‐COSY, MLEV17 HOHAHA and NOESY techniques. Interproton nuclear Overhauser effects, spin‐spin coupling constant of vicinal H‐NCα‐H protons and deuterium exchange rates of individual NH groups were derived from two‐dimensional NMR spectra. The data unequivocally define the peptide conformation as the right‐handed α‐helix, extremely rigid in the central region from Phe 42 to Nle 60 and flexible in the N‐ and C‐terminal parts.
α‐Toxin binding to acetylcholine receptor α179–191 peptides: Intrinsic fluorescence studies
Authors: Radding W. , Corfield P.W.R. , Levinson L.S. , Hashim G.A. , Low B.W.
Published: July 2002

Interactions between two α‐toxins and the synthetic peptides α179–191 from both calf and human acetylcholine receptor α‐subunit sequences have been studied by measurements of quenching of intrinsic fluorescence after toxin addition. Dissociation constants of approx. 5 × 10−8 M for binding of calf peptide by both α‐cobratoxin and erabutoxin a have been estimated. The binding of α‐cobratoxin to calf peptide, which leads to marked quenching of fluorescence intensity, is inhibited by a 104 molar excess of acetylcholine. The human α179–191 peptide binds to α‐cobratoxin, but not, under comparable conditions, to erabutoxin a.
Mitochondrial protein import: Differential recognition of various transport intermediates by antibodies
Authors: Söllner Thomas , Pfanner Nikolaus , Neupert Walter
Published: July 2002

The precursors of the mitochondrial proteins ADP/ATP carrier (AAC) and F1‐ATPase subunit β (F1β) were accumulated at the stages of binding to receptor sites on the mitochondrial outer membrane, or in contact sites between outer and inner membranes. Specific antibodies raised against the mature proteins were added to the isolated mitochondria and efficiently bound to these translocation intermediates. Further movement of the precursors to consecutive steps along their import pathway was thereby inhibited. Controls showed that precursor proteins which were inserted into or translocated across the outer membrane were not recognized by the antibodies unless the mitochondrial membranes were disrupted. We conclude that the trapped translocation intermediates have antigenic sites exposed to the outside of the outer membrane.
A model for the molecular mechanism of interfacial activation of phospholipase A2 supporting the substrate theory
Authors: Thuren Tom
Published: July 2002

Changes occurring in the activity of porcine pancreatic phospholipase A2 upon formation of mixed micelles of sodium cholate and the fluorescent phosphocholines 1,2‐di [6‐(pyren‐1‐yl)butanoyl]‐sn‐glycero‐3‐phosphocholine or 1‐[6‐(pyren‐1‐yl)butanoyl]‐2‐[6‐(pyren‐1‐yl)hexanoyl]‐sn ‐glycero‐3‐phosphocholine were studied. A 2‐fold enhancement was observed in the activity of phospholipase A2 towards both pyrene phospholipids upon exceeding the critical micellar concentration of the system. Changes in the pyrene excimer/monomer fluorescence emission intensity ratio coincide with the enhancement of phospholipase A2 activity at the critical micellar concentration. Due to the different effects of micellization on the alignment of the pyrene in the two fluorescent probes conformational changes could be assessed. A model describing possible conformations of these pyrene phospholipid molecules below and above the critical micellar concentration is presented and correlated with the interfacial activation of phospholipase A2.
Phosphatidylinositol turnover is not a general regulator of neuroblastoma cell differentiation: comparison between two differentiating agents, retinoic acid and γ‐interferon
Authors: Lanciotti M. , Cornaglia-Ferraris P. , Ponzoni M.
Published: July 2002

The turnover of phosphatidylinositol (PI) is believed to constitute a crucial step in the signaling pathways for stimulation of cells by a variety of bioactive substances, including differentiating agents; however decisive evidence for the idea has not been obtained. In the present paper, we investigated the involvement of PI turnover in cell differentiation using a human neuroblastoma cell line, LAN‐1, which can be induced to differentiate along the neuronal pathway by both retinoic acid (RA) and γ‐interferon (γ‐IFN). Analysis of labelled phosphatidylinositol metabolises from prelabelled cells indicated a rapid decrease of inositol 1,4,5‐trisphosphate and 1,2‐diacylglycerol within 1 min of induction of LAN‐1 cell dilfferentiation by RA, while no changes were observed in γ‐IFN‐treated cells. These findings indicate the occurrence of decreased inositol phospholipid turnover in RA‐treated LAN‐1 cells and suggest that phosphoinositide‐derived metabolises may not constitute general regulators of cellular differentiation.
Okadaic acid, a specific protein phosphatase inhibitor, induces maturation and MPF formation in Xenopus laevis oocytes
Authors: Goris Jozef , Hermann Jacques , Hendrix Peter , Ozon René , Merlevede Wilfried
Published: July 2002

Micro‐injection of, or incubation with okadaic acid (OA), a specific phosphatase inhibitor, can induce formation of maturation‐promoting factor (MPF) and germinal vesicle breakdown (GVBD) in Xenopus laevis oocytes. Comparison of the dose‐response curves of OA on maturation, isolated enzymes and phosphatase activities in crude oocyte preparations suggests that inhibition of both polycation‐stimulated (PCS) and ATP,Mg‐dependent (AMD) phosphatases is sufficient but requires that a critical phosphorylation level is attained of one or several of their substrates, resulting in the formation of active MPF and meiotic maturation.
Level of protein kinase C activity correlates directly with resistance to adriamycin in murine fibrosarcoma cells
Authors: O'Brian Catherine A. , Fan Dominic , Ward Nancy E. , Seid Christopher , Fidler Isaiah J.
Published: July 2002

In this report, we demonstrate a direct correlation between protein kinase C (PKC) activity and adriamycin (ADR) resistance in mouse fibrosarcoma cells. PKC activity was measured in four murine UV‐2237M fibrosarcoma cell lines that differed in the degrees to which they expressed resistance to ADR, which is an inhibitor of PKC. A comparison of the four cell lines revealed a positive correlation between the level of PKC activity and resistance to ADR. Incubation of the cells with the PKC inhibitor H‐7 produced a partial reversal of ADR resistance. Taken together, these results suggest a role for PKC in the mechanism of ADR resistance.
Quantitative immunochemical evidence for identical topography of subunits CF0II and CF0I within the photosynthetic ATP‐synthase of spinach chloroplasts
Authors: Otto Joachim , Berzborn Richard J.
Published: July 2002

Monospecific polyclonal antisera against the 16 kDa and the 18 kDa polypeptides II and I from the CF0 moiety of the photosynthetic ATP‐synthase in spinach were produced. Inhibition, agglutination and absorption experiments were combined with quantitative determinations in ELISA of residual antibodies and of amounts of CF0 and CF1. It follows that subunits CF0II and CF0I are membrane anchored, extend into the matrix space, and are partially shielded underneath or within CF1. These results on the structure suggest that CF0II and CF0I are homologous to each other, and in function both homologous to E. coli F0b; thus CF0II together with CF0I would be part of the ‘stalk’.
P2‐purinergic agonists activate phospholipase C in a guanine nucleotide‐ and Ca2+‐dependent manner in FRTL‐5 thyroid cell membranes
Authors: Okajima Fumikazu , Sato Koichi , Kondo Yoichi
Published: July 2002

Various adenine nucleotides activated phospholipase C of FRTL‐5 cell membranes in the following order of activity, ATPγS > ATP > AppNp > AppCp = ADP > MeSATP. This order was well consistent with that observed in intact cells. Such activation occurred only in the presence of appropriate concentrations of GTPγS and Ca2+, in a way similar to the norepinephrine‐induced activation. NaF, a non‐specific GTP‐binding protein (G‐protein) activator, also stimulated the enzyme. These adenine nucleotides, norepinephrine and NaF‐induced activations were inhibited by GDPβS. We conclude that a G‐protein is involved in the adenine nucleotides‐induced activation of phospholipase C via P2‐purinergic receptor in FRTL‐5 cells.
The interaction of alkynyl carboxylates with serine enzymes a potent new class of serine enzyme inhibitors
Authors: Segal Dina , Shalitin Yechiel , Wingert Horst , Kitamura Tsugio , Stang Peter J.
Published: July 2002

The recently reported alkynyl esters, propynyl benzoate and propynyl p‐methoxybenzoate, were found to interact with a variety of serine enzymes. α‐Chymotrypsin was inhibited very rapidly by an equivalent amount of the esters. Trypsin, elastase and pronase were also inhibited by the esters. On the other hand, liver esterase started to hydrolyse the alkynyl esters rapidly, but the enzyme became inhibited during the course of reaction. The inhibited enzymes exhibited slow reactivation which could be considerably enhanced by hydroxylamine.
Secretory protein synthesis in Chironomus salivary gland cells is not coupled with protein translocation across endoplasmic reticulum membranes
Authors: Kiseleva E.V.
Published: July 2002

Fragments of rough endoplasmic reticulum containing polysomes bound to the membranes only at the 5' end were visualized in electron microscopic spreads from Chironomus thummi salivary gland cells. The length of the nascent protein molecules in the polysemes increased from the 5' to the 3' (free) polysome end. The data obtained disagree with the generally accepted model according to which synthesis of secretory proteins is concomitant with the protein transport across the endoplasmic membrane
Variable number of repeat units in genes encoding Trypanosoma cruzi antigens
Authors: Macina Roberto A. , Affranchino José L. , Pollevick Guido D. , Jazin Elena E. , Frasch Alberto C.C.
Published: July 2002

The genes encoding a protein antigenic during the acute phase (SAPA) and another one antigenic during the chronic phase (antigen 30) of human Chagas' disease were analyzed in 15 Trypanosoma cruzi isolates and clones collected in distant geographical regions. These two genes had tandem repeats which were present in all parasites tested. However, large differences in the length of restriction fragments were observed among isolates. This was readily explained by variations in the number of repeat units present in homologous genes. This result was confirmed after analysis of 3 members of the SAPA gene family. In the case of antigen 30, we propose that differences in the number of repeat units result in size differences in the corresponding RNAs and proteins, which explains the large size heterogeneity in otherwise homologous T. cruzi antigens.
Putative N‐terminal splitting enzyme of amyloid A4 peptides is the multicatalytic proteinase, ingensin, which is widely distributed in mammalian cells
Authors: Ishiura Shoichi , Tsukahara Toshifumi , Tabira Takeshi , Sugita Hideo
Published: July 2002

The main characteristic changes observed in Alzheimer's disease (AD) are the presence of neurofibrillary tangles and the deposition of amyloid A4 peptides. The most abundant amyloid A4 peptide species in AD (which we tentatively named A4') is composed of 39 amino acids, which is devoid of the 3 N‐terminal amino acids, Asp‐Ala‐Glu, of the originally reported A4 peptide. We synthesized a model peptide substrate, Suc‐Ala‐Glu‐methylcoumarinamide (MCA), to identify the proteinase that splits the A4' peptide. DEAE‐cellulose column chromatography of rat liver and porcine brain extracts showed that only one peak material digested the synthetic substrate at pH 8. The results for the final preparation indicate that the Suc‐Ala‐Glu‐MCA‐degrading enzyme is a high‐molecular‐mass proteinase, with a molecular mass of above 500 000, and is composed of several low‐molecular‐mass subunits. These results suggest that a non‐lysosomal multicatalytic proteinase (we named this enzyme ingensin (ingens = large in Latin), Ishiura, S. et al. (1985) FEBS Lett. 189, 119‐123) catalyzes the above reaction. Antiserum against the purified multicatalytic proteinase, ingensin, crossreacted with the purified Suc‐Ala‐Glu‐MCA‐degrading proteinase. It is likely that ingensin shows a similar action toward amyloid precursor protein (APP) in vivo.
High molecular weight forms of basic fibroblast growth factor recognized by a new anti‐bFGF antibody
Authors: Grothe Claudia , Zachmann Karolin , Unsicker Klaus , Westermann Reiner
Published: July 2002

An antibody against basic fibroblast growth factor (bFGF) was raised using purified bovine pituitary bFGF. Western blot analysis revealed immunoreactive bands at 18, 24, 30–33 and 46 kDa in inununoaffinity purified extracts of pituitary and adrenal gland using this antibody. A similar staining pattern was obtained with ovary extracts with the exception of the missing 18 kDa band. A second anti‐bFGF antibody raised against a synthetic peptide comprising the 24 N‐terminal amino acids of bFGF reacted with the 18 kDa and the 46 kDa band of immunoaffinity purified ovary and adrenal gland extracts.
Shape changes of the human red cell studied by aqueous two‐phase partition
Authors: Backman Lars
Published: July 2002

Aqueous two‐phase systems have been used to study the human red cell during metabolically induced shape changes. When the discoid character of the cells was lost in favour of echinocytic forms, the partition increased both in charge‐sensitive and in charge‐insensitive two‐phase systems. Reversal of the shape transformation by ATP repletion not only led to shape recovery but also restored the initial partition. Therefore it is apparent that red cells exhibit a shape‐dependent partition behaviour. As the partition is dependent on surface properties (such as charge and hydrophobicity) of the partitioned material, the results show that the shape changes caused rearrangement of the membrane and thereby exposure of or greater accessibility of binding groups on the cell surface. The similar partition behaviour in the charge‐sensitive and charge‐insensitive phase systems show that the increased partition was caused mainly by increased hydrophobic interactions between the cells and the upper phase. The observed partition behaviour therefore suggests that the echinocytic cells acquire a higher affinity for the upper phase by repacking the lipid bilayer or at least the outer leaflet into a less efficient packed and thus more fluid structure.
Multiple mechanisms of protein kinase C inhibition by triphenylacrylonitrile antiestrogens
Authors: Bignon Eric , Pons Michel , Gilbert Jacques , Nishizuka Yasutomi
Published: July 2002

The activation of type I (γ), II (β) and III (α) protein kinase C (PKC) subspecies by phosphatidylserine (PS) and diacylglycerol (DAG) is inhibited by micromolar concentrations of triphenylacrylonitrile (TPE) antiestrogens. TPE A (with p‐hydroxy and p‐diethylaminoethoxy groups on the 3‐and 3'‐phenyl rings, respectively) interacts with PS‐vesicles as well as with the regulatory domain of PKC, probably at a site different from the Ca2+ and DAG binding sites. The interaction of TPE A with the regulatory domain of enzyme is very slow. Apparently, TPE A does not interact with the catalytic domain of PKC. In contrast, another TPE derivative, TPE B (with a p‐hydroxy group on each of the three phenyl rings) inhibits the enzyme activity in a competitive manner with respect to ATP, suggesting that this TPE interacts with the catalytically active site of the enzyme. It seems likely that various TPE antiestrogen derivatives may exert their inhibitory action on PKC by multiple different mechanisms.
CD4‐like molecules in human sperm
Authors: Gobert Bernard , Amiel Corinne , Tang Jianqing , Barbarino Patricia , Christine Béné Marie , Faure Gilbert
Published: July 2002

The expression of a molecule recognized by CD4 monoclonal antibodies was investigated on human sperm using immunolabelling, biochemical and immunochemical methods. Flow cytometry detected a significant fluorescence signal. SDS‐PAGE analysis and Western blotting identified a molecule of 60 kDa, consistent with a CD4‐like structure as confirmed after selective immunoseparation. Additional bands reacting with anti‐CD4 were found in sperm extracts (73 kDa) and seminal fluid (90 kDa). These data indicate that sperm express a molecule similar to the receptor for HIV described on mononuclear cells.
Analogs of human epidermal growth factor which partially inhibit the growth factor‐dependent protein‐tyrosine kinase activity of the epidermal growth factor receptor
Authors: Matsunami Risë K. , Campion Stephen R. , Niyogi Salil K. , Stevens Audrey
Published: July 2002

Three site‐directed mutants of human epidermal growth factor, Leu‐26 → Gly, Leu‐47 → Ala, and He‐23 → Thr, were examined for their ability to stimulate the protein‐tyrosine kinase activity of the epidermal growth factor receptor. The receptor binding affinities of the mutant growth factors were 20‐ to 50‐fold lower, as compared to wild‐type growth factor. At saturating concentrations of growth factor, the velocities of the phosphorylation of exogenously added substrate and receptor autophosphorylation were significantly lower with the mutant analogs, suggesting a partial ‘uncoupling’ of signal transduction. The mutant analogs were shown to compete directly with the binding of wild‐type, resulting in a decrease in growth factor‐stimulated kinase activity.
Photoaffinity labeling of the lysosomal neuraminidase from bovine testis
Authors: van der Horst Gijsbertus T.J. , Rose Ursula , Brossmer Reinhard , Verheijen Frans W.
Published: July 2002

ASA‐NeuAc2en, a photoreactive arylazide derivative of sialic acid, is shown to be a powerful competitive inhibitor of lysosomal neuraminidase from bovine testis (K
i ≈ 21 μM). Photoaffinity labeling and partial purification of preparations containing this lysosomal neuraminidase activity result in specifically and non‐specifically labeled polypeptides. Only labeling in a 55 kDa polypeptide is found to be specific, since it could be prevented by the competitive neuraminidase inhibitor NeuAc2en. We conclude that the 55 kDa polypeptide in the bovine testis β‐galactosidase/neuraminidase/protective protein complex contains the catalytic site of neuraminidase.
Pressure‐induced dissociation of tight couple ribosomes
Authors: Groß Michael , Jaenicke Rainer
Published: July 2002

Ribosomes from Escherichia coli have been shown to undergo subunit dissociation at elevated hydrostatic pressure. This holds for both crude and highly purified ribosomes. No inhibitory effect could be detected by addition of either the S100 supernatant, or tRNA, polyuridylic acid, and spermine. Light scattering experiments at pressures up to 1000 bar reveal different susceptibility of tight couple and loose couple ribosomes toward pressure dissociation. Tight couples are subjected to EF‐Tu‐catalyzed binding of aminoacyl‐tRNA, thus yielding a model system of the elongating ribosome before the peptidyl transfer step. High pressure dissociation of this compound suggests that enzymatic binding converts tight couples into loose couples. A hypothesis referring to conformational changes during the elongation cycle is presented.
Regulation of the level of uncoupling protein in brown adipose tissue by insulin requires the mediation of the sympathetic nervous system
Authors: Géloën Alain , Trayhurn Paul
Published: July 2002

The role of the sympathetic nervous system in the regulation by insulin of the level of uncoupling protein in brown adipose tissue has been examined. The amount of uncoupling protein was substantially reduced in streptozotocin‐diabetic rats, while insulin replacement to diabetic animals induced a partial restoration. Unilateral denervation of the interscapular brown fat pads also lowered the amount of uncoupling protein, and in diabetic animals inhibited the stimulation of the level of the protein by insulin replacement. Maintenance of normal uncoupling protein levels requires both insulin and the sympathetic system; regulation of the protein by insulin involves sympathetic mediation.
Complementation of the protein transport defect of an Escherichia coli sec Y mutant (sec Y24) by Bacillus subtilis sec Y homologue
Authors: Nakamura Kouji , Takamatsu Hiromu , Akiyama Yoshinori , Ito Koreaki , Yamane Kunio
Published: July 2002

Bacillus subtilis SecY homologue shares 41.3% homology with that of E. coli and remarkably higher homologous regions (more than 80%) are present in the four cytoplasmic regions [(1990) J. Biochem. 107, 603‐607]. Based on the formation of the mature form of OmpA in E. coli, we have shown that the protein transport defect of the E. coli sec Y mutant (secY24) is complemented by the gene product from the B. subtilis sec Y homologue, which is expressed under the lac promoter control. However, B. subtilis SecY could not restore growth of the E. coli mutant at non‐permissive temperature.
Reversible thermal unfolding of Bacillus subtilis levansucrase is modulated by Fe3+ and Ca2+
Authors: Chambert Régis , Petit-Glatron Marie-Françoise
Published: July 2002

The equilibrium transition curves for thermal unfolding of levansucrase were established at several pH values. At pH 7 and within the temperature range of bacterial growth, the unfolded from is predominant. However, under such conditions, refolding is promoted by the only addition of Ca2+ or Fe3+. We propose that the tertiary structure flexibility of levansucrase plays a key role in its secretion process.
Cytochrome P‐450LM2 oligomers in proteoliposomes
Authors: Myasoedova Ksenia N. , Berndt Peter
Published: July 2002

The effects of bifunctional cross‐linking reagents on the purified hexameric cytochrome P‐450LM2 in an aqueous medium and on the proteoliposomal cytochrome P‐450LM2 have been compared. In both cases, cross‐linking is shown to result in the appearance of a range of additional protein bands in SDS electrophoretograms. The number and the position of these bands seem to be similar in the solubilized and in the proteoliposomal cytochromes. No additional bands appear when the purified cytochrome P‐450 was pretreated with ?2? Emulgen 913, which decomposes cytochrome P‐450LM2 hexamers. The results indicate that the membrane‐bound cytochrome P‐450 can exist in the oligomeric (presumably hexametric) form.
[3H]Morphine binding is enhanced by IL‐1‐stimulated thymocyte proliferation
Authors: Roy Sabita , Ge Bang-Lun , Ramakrishnan S. , Lee Nancy M. , Loh Horace H.
Published: July 2002

Mouse thymocytes incubated in vitro with increasing concentrations of interleukin‐1 (IL‐1) in the presence of phytohemagglutinin (PHA) exhibited a dose‐dependent increase in cell proliferation, as measured by [3H]thymidine incorporation. Under these conditions, there was a parallel dosedependent increase in specific [3H]morphine binding, with a maximum increase of approximately 5‐fold over basal levels. The binding sites differ from classical opioid receptors in that they are not stereo‐selective. Interleukin‐2 was ineffective in promoting either cell proliferation or enhanced opioid binding, but the effects of IL‐1 could be mimicked by phorbol myristate acetate (PMA), suggesting the involvement of tyrosine phosphorylation. These results indicate that morphine‐binding sites on immune cells can be regulated by cytokine activation.
Isolation and biochemical characterization of a zona pellucida‐binding glycoprotein of boar spermatozoa
Authors: Jonáková Vèra , Sanz Libia , Calvete Juan J. , Henschen Agnes , Čechová Dana , Töpfer-Petersen Edda
Published: July 2002

Lectin‐like molecules on the sperm surface are implicated in the process of gamete recognition and adhesion. We have isolated and biochemically characterized a 15 kDa glycoprotein from ejaculated boar sperm which possess zona pellucida‐binding‐ and haemagglutinating‐activity. The zona/ 15 kDa protein interaction is inhibited by fucoldan, suggesting that the glycoprotein is one of the sperm components which participate in the initial gamete interaction. N‐Terminal sequence analysis of the isolated 15 kDa glycoprotein showed that it may belong to the same sperm/egg recognition‐mediating protein family as the sea urchin sperm protein binding.
Diversity of the protein kinase gene family in rice
Authors: Feng Xin-Hua , Kung Shain-dow
Published: July 2002

Multiple genes have been found to encode families of protein kinases in animals and yeasts. Little is known of the diversity of protein kinase families in plants. We have used the polymerase chain reaction to identify members of protein kinase gene family in rice. We have cloned eight partial cDNA sequences from which deduced amino acid sequences contained conserved sequences or amino acid residues characteristic of catalytic domains of eukaryotic protein serine/threonine kinases. Our results suggest that there is great complexity in the protein kinase gene family in plants and that protein phophorylation may play an as important role in plants as in other eukaryotes.
Isolation of a novel arachidonic acid metabolite 3‐hydroxy‐5,8,11,14‐eicosatetraenoic acid (3‐HETE) from the yeast Dipodascopsis uninucleata UOFs‐Y128
Authors: van Dyk M.S. , Kock J.L.F. , Coetzee D.J. , Augustyn O.P.H. , Nigam S.
Published: July 2002

When arachidonic acid (AA) is added to the yeast Dipodascopsis uninucleata UOFS Y128, one of the major metabolites isolated and purified with the help of thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) is 3‐hydroxy‐5,8,11,14‐eicosatetraenoic acid (3‐HETE). The structure of this new AA metabolite was elucidated mainly by electron impact (EI) mass spectrometry (MS). Strikingly, the formation of this new metabolite was found to be inhibited by aspirin.
Nucleotide sequences of the continuous and separated petA, petB and petD chloroplast genes in Chlamydomonas reinhardtii
Authors: Büschlen S. , Choquet Y. , Kuras R. , Wollman F.-A.
Published: July 2002

We have mapped and sequenced the petA (cytf), petB (cytb6) and petD (subunit IV) genes on the chloroplast genome of Chlamydomonas reinhardtii. At variance with the pet genes in higher plant chloroplasts, the petB and petD genes are continuous, not adjacent and not located next to the psbB gene. The corresponding polypeptide sequences are highly conserved when compared with their counterparts from other sources but have a few features specific of algal cytb6/f complexes, In particular the transit sequence of cytf displays unique characteristics when compared with those previously described for cytf in higher plants.
The induction of adipose conversion in 3T3‐L1 cells is associated with early phosphorylation of a 60‐kDa nuclear protein
Authors: Brandes Ruth , Arad Rivka , Bar-Tana Jacob
Published: July 2002

The induction of adipose conversion in 3T3‐L1 cells by bezafibrate has been shown to be enhanced by dibutyryl‐cAMP. We here report that the induction of adipose conversion in 3T3‐L1 cells, by either bezafibrate and dibutyryl cAMP, or isobutylmethyxanthine alone, is associated with a very early phosphorylation of a 60‐kDa acidic protein found in the nuclear fraction.
Involvement of sapecin in embryonic cell proliferation of Sarcophaga peregrina (flesh fly)
Authors: Komano Hiroto , Homma Ko-ichi , Natori Shunji
Published: July 2002

Addition of antibodies against sapecin to the culture medium of NIH‐Sape‐4 cells derived from a Sarcophaga embryo greatly inhibited cell proliferation, whereas addition of sapecin stimulated cell proliferation. These results suggest that sapecin is involved in the proliferation of embryonic cells of Sarcophaga. Sapecin is known to have potent antibacterial activity, so it seems to have two different biological functions: i.e. protection against bacterial infection and stimulation of embryonic cell proliferation.
Comment to Shinohara et al. (1991) FEBS Letters 293, 1731̄74 The uncoupling protein is not expressed in rat liver
Authors: Ricquier Daniel , Raimbault Serge , Champigny Odette , Miroux Bruno , Bouillaud Frédéric
Published: July 2002

Using Northern blot analysis, immunoblotting with purified antibodies and Polymerase Chain Reaction analysis, we were unable to detect the Uncoupling Protein‐UCP or its mRNA in liver of control, cold‐exposed or newborn rats. The unique expression of this protein in brown adipocytes was confirmed. These data refute the surprising recent report on UCP expression in rat liver (Shinohara (1991) FEBS Lett. 293, 173–174). Moreover we report that the hybridization signal obtained by these authors is probably non‐specific and due to the 3′ non‐coding domain of the UCP cDNA probe.
Functional high efficiency expression of cloned leucocyte chemoattractant receptor cDNAs
Authors: Didsbury Jolin R. , Uhing Ronald J. , Tomhave Eric , Gerard Craig , Gerard Norma , Snyderman Ralph
Published: July 2002

Human kidney 293 TSA cells were transfected by a calcium phosphate method with human formylpeptide and C5a receptor cDNAs with high efficiency. Formylpeptide receptor positive transfectants expressed a total of 968,000 ± 34,000 receptors per cell with two affinity states (K
ds of ca. 0.43 nM and 39 nM), which in the presence of 100μM GTPγS decreased by ca. 4‐fold the number of high‐affinity sites. The ligand binding pharmacology of cloned and expressed formylpeptide receptors were indistinguishable from endogenous receptors on human neutrophils. Expressed formylpeptide and C5a receptors were functionally active in mobilizing intracellular calcium via a pertussis toxin sensitive mechanism with an ED50 for formylpeptide of ca. 0.5–1.0 nM. This expression system, in which receptor expression can be monitored by flow cytometric methods and in which intracellular calcium responses are measurable, unlike in the more popular COS‐7 cell expression system, will provide a useful basis for the analysis of chemoattractant receptor structure‐function relationships.
Protein kinase Cζ and n in murine epidermis TPA induces down‐regulation of PKCn but not PKCζ
Authors: Gschwendt Michael , Leibersperger Hanno , Kittstein Walter , Marks Friedrich
Published: July 2002

Murine epidermis contains PKCζ and n as evidenced by the application or specific antisera. PKCζ predominates in the cytosol and PKCn in the particulate fraction. PKCζ is shown to be present also in other murine tissues, with large amounts found in lung. Whereas epidermal PKCn is completely down‐regulated by treatment or mouse skin with TPA or bryostatin 1 for 18 h. PKCζ is neither translocated by treatment with TPA for 20 min, nor down‐regulated by treatment with TPA or bryostatin 1 for 18 h. PKCζ is activated by phosphatidyl serine alone and does neither respond to Ca2+ nor to TPA. It is inhibited by staurosporine with an IC50 of 16 nM, which is within the same range or other PKC isoenzymes. The sensitivity of PKCζ towards the staurosporine derivative K252a is similar to that or PKCα,β,γ but much higher than that of PKCδ and ε.
Intracellular signal transduction pathways that control pancreatic β‐cell proliferation
Authors: Sjöholm Åke
Published: July 2002

This review focuses on the factors that regulate the proliferation of pancreatic islet β‐cells in vitro, and in particular on the intracellular pathways that convey the mitogenic signal into a proliferative response. Substances as diverse as nutrients, polypeptides, cytokines, adrenergic agents, lithium, phorbol esters and cyclic AMP analogs are all able to stimulate or inhibit β‐cell proliferation in a time‐ and concentration‐dependent manner. The evidence for involvement of cyclic AMP, cyclic GMP, protein kinase C, inositol polyphosphates, GTP‐binding proteins, polyamines and oncogenes is reviewed.
Interdependence of respiratory NO reduction and nitrite reduction revealed by mutagenesis of nirQ, a novel gene in the denitrification gene cluster of Pseudomonas stutzeri
Authors: Jüngst Angelika , Zumft Walter G.
Published: July 2002

An open reading frame, designated nirQ, was identified upstream or nirS, the structural gene for the respiratory nitrite reductase or Pseudomonas stutzeri ZoBell. Its derived gene product (275 amino acids, M
r = 30.544) shows similarity to the NtrC protein family or transcriptional activators. Deletion—replacement mutagenesis of the nirQ gene resulted in the simultaneous loss of nitrite reduction and NO reduction in vivo. However, both reductases were still synthesized, with only nitrite reductase being active in vitro. NO reductase was overproduced by a factor of about 2. Our results indicate that the systems for nitrite reduction and NO reduction are functionally coupled.
Tumor necrosis factor modulation of expression of the cystic fibrosis transmembrane conductance regulator gene
Authors: Nakamura Hidenori , Yoshimura Kunihiko , Bajocchi Gianluigi , Trapnell Bruce C. , Pavirani Andrea , Crystal Ronald G.
Published: July 2002

Based on the knowledge that expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene can be modulated at the transcriptional level, and that the CFTR gene promoter contains sequences homologous to elements in other promoters that respond to tumor necrosis factor‐α (TNF), we evaluated the hypothesis that TNF might modulate CFTR gene expression in epithelial cells. Studies with HT‐29 cells, a colon epithelium‐derived tumor cell line known to express the CFTR gene, demonstrated that TNF downregulated CFTR mRNA transcript levels in a dose‐ and time‐dependent fashion. Interestingly, nuclear run‐on analyses demonstrated that TNF did not affect the rate of transcription of CFTR gene, but exposure of the cells to TNF did modify the stability of CFTR mRNA transcripts, resulting in a mRNA half‐life that was reduced to 65% of the resting level. These observations suggest that CFTR gene expression can be modulated by TNF, at least in part, at the post‐transcriptional level.
A 5 kDa protein (SCS23) from the 30 S subunit of the spinach chloroplast ribosome
Authors: Wada Akira , Koyama Kazunari , Maki Yasushi , Shimoi Yukihiro , Tanaka Ayumi , Tsuji Hideo
Published: July 2002

The proteins of the 30 S ribosomal subunits from spinach chloroplasts were investigated using a radical‐free and highly reducing (RFHR) method of two‐dimensional polyacrylamide gel electrophoresis (PAGE). Twenty‐three proteins were resolved on the gel down to the smallest protein of 5 kDa. The N‐terminal amino acid sequence of the 5 kDa protein showed no homology with that of any other protein stored in databases, and the copy numbers were estimated to be 0.88±0.16 and 0.72±0.04 in the 30 S subunits and the 70 S ribosomes, respectively. The results suggest that the 5 kDa protein, which we have called SCS23, may be an essential ribosomal protein specific to spinach chloroplasts.
Assignment of the ligand binding site of the porcine estradiol receptor to the N‐terminal 17 kDa part of domain E
Authors: Thole Hubert H.
Published: July 2002

Ligand‐filled porcine estradiol receptor was adsorbed to heparin‐Sepharose, from which a 26 kDa fragment was released by papain. Its mass was reduced to a 17 kDa fragment by trypsin. The sedimentation velocities of both estradiol binding fragments increased after reaction with the MAB 13H2. The same antibody identified the denatured fragments on Western blots. The N‐terminal 21 amino acid sequence was obtained from the 17 kDa peptide which corresponds to amino acids 303–323 of the human receptor with four amino acids exchanged. This indicates that the ligand binding site resides in the N‐terminal 17 kDa portion of domain E.
Thrombospondin (TSP1) mediates in vitro proliferation of human MG‐63 osteoblastic cells induced by α‐thrombin
Authors: Abbadia Zoher , Clezardin Philippe , Serre Claire-Marie , Amiral Jean , Delmas Pierre D.
Published: July 2002

Thrombospondin (TSP) is a 450‐kDa glycoprotein synthesized and secreted by human MG‐63 osteoblastic cells. In this study, we have first studied the effect of α‐thrombin on TSP expression by human MG‐63 cells. In situ hybridization indicated that TSP mRNA level in thrombin‐treated MG‐63 cells was increased when compared to unstimulated cells. As judged by immunofluorescence, thrombin‐treatment of MG‐63 cells resulted in increased cell surface expression of TSP when compared to quiescent cells. Because thrombin stimulates proliferation of osteoblastic cells, the involvement of TSP in proliferation of thrombin‐stimulated osteoblastic cells was then investigated using a serum‐free mitogenesis assay. Both α‐thrombin (0.01 to 0.15 U/ml) and TSP (5 to 600 ng/ml) caused a dose‐dependent increase in [3H]thymidine incorporation by MG‐63 cells. Proliferation of osteoblastic cells induced by α‐thrombin or TSP was specifically and totally inhibited by anti‐TSP monoclonal antibodies (3–10 μg/ml) or by indomethacin (1 μM), an inhibitor of prostaglandin synthesis. Anti‐TSP antibodies which inhibited cell proliferation also inhibit TSP expression to the surface of these cells. Our experiments support the existence of a mechanism whereby TSP bound to the cell surface of thrombin‐treated MG‐63 cells stimulates secretion of prostaglandins which, in turn, allow cell proliferation to proceed.
Enzymes of the cyclic GMP metabolism in bovine retina
Authors: Gaidarov I.O. , Suslov O.N. , Abdulaev N.G.
Published: July 2002

Guanylate kinase (EC 2.7.4.8) catalyzing the reaction GMP + ATP = GDP + ADP, was purified to homogeneity from bovine retina. Using oligonucleotides based on the amino acid sequence of this enzyme, the cDNA encoding guanylate kinase (GK) was isolated and its nucleotide sequence was determined. Expression of the GK cDNA in E. coli, and the purification and functional characterization of the expressed enzyme are presented. It is shown that bovine retinal GK, like its yeast counterpart, contains the characteristic glycine‐rich motif and all the amino acids involved in GMP binding. Bovine retinal enzyme is extended for several amino acid residues both at the N‐ and C‐termini, compared to the yeast enzyme.
Purification and characterization of the trefoil peptide human spasmolytic polypeptide (hSP) produced in yeast
Authors: Thim Lars , Norris Kjeld , Norris Fanny , Nielsen Per F. , Bjørn Søren E. , Christensen Mogens , Petersen Jørgen
Published: July 2002

Recombinant human spasmolytic polypeptide (r‐hSP) has been produced in relatively large amounts in Saccharomyces cerevisiae. The two intronless trefoil domains of the hSP‐DNA were cloned separately by PCR from human genomic DNA, and the remaining parts of the gene synthezised. Recombinant plasmids were constructed to encode a fusion protein consisting of a hybrid leader sequence and the hSP sequence. The leader sequence serves to direct the fusion protein into the secretory pathway of the cell and to expose it to the Kex 2 processing enzyme system. The secreted r‐hSP was found in a glycosylated and an non‐glycosylated form. The two forms of r‐hSP were purified from the yeast fermentation broth by a combination of ion‐exchange chromatography and preparative HPLC. The overall yield from 8 litres of fermentation broth was 160 mg r‐hSP and 219 mg glycosylated r‐hSP corresponding to 50% and 34%, respectively. The structure of the r‐hSP and the glycosylated r‐hSP was determined by amino acid analysis and carbohydrate composition analysis as well as by peptide mapping, amino acid sequencing and mass spectrometric analysis.
The outer membrane of plant mitochondria contains a calcium‐dependent protein kinase and multiple phosphoproteins
Authors: Pical Christophe , Fredlund Kenneth M. , Petit Patrice X. , Sommarin Marianne , Møller lan M.
Published: July 2002

Highly purified mitochondria from potato (Solomon tuberosum L. cv. Bintje) tubers were subfractionated into a matrix fraction, an inner membrane fraction and an outer membrane fraction with minimal cross‐contamination. When the matrix and inner membrane fractions were incubated with [γ‐32P]ATP only one and three prominent phosphoproteins were detected after SDS‐PAGE and autoradiography, respectively. In contrast, more than 20 phosphoproteins could be labelled in the outer membrane fraction, the main ones at 12, 18, 26, 43, 58, 60, 65, 74 and 110 kDa. Only one band, at 18 kDa, was detectable when the labelling was done in the presence of EGTA. We conclude that the outer membrane of plant mitochondria contains at least one Ca2+‐dependent protein kinase and more than 20 endogenous substrates.
Expression of highly active sex‐inducing pheromone of Volvox carteri f. nagariensis in a mammalian cell system
Authors: Jaenicke Lothar , Feldwisch Ortrun , Merkl Barbara , Cremer Anja , Haas Ingrid G.
Published: July 2002

A cDNA fragment coding for the sex‐inducing glycoprotein of Volvox carteri f. nagariensis was expressed in a mammalian cell system (baby hamster kidney (BHK) cells). The transfection product exhibited a specific biological activity intermediate between the natural pheromone of the strains Volvox carteri f. nagariensis and Volvox carteri f. weismannia. Immunoblot analysis showed that the sex‐inducing activity was expressed as a set of three iso‐glycoproteins (35, 34 and 31 kDa).
RNA editing in trypanosome mitochondria: guidelines for models
Authors: Sloof Paul , Benne Rob
Published: July 2002

Mitochondrial RNAs in trypanosomes are post‐transcriptionally altered by uridine insertion and deletion. The information for these RNA editing processes, which are essential for the production of functional messengers, is provided by small guide RNAs. This article discusses how features of partially edited RNAs, gRNAs and chimeric RNAs, in which a gRNA is covalently linked to an editing site of pre‐mRNA, have been used for the construction of models.
Association of three small GTP‐binding proteins with cholinergic synaptic vesicles
Authors: Volknandt Walter , Pevsne Jonathan , Elferink Lisa A. , Scheller Richard H.
Published: July 2002

Several small (low molecular weight) GTP‐binding proteins are associated with cholinergic synaptic vesicles derived from the electric organ of electric ray. Using GTP overlay techniques and direct micro sequencing we analyzed the association of small GTP‐binding proteins with synaptic vesicles. Both experimental procedures revealed the specific occurrence of multiple small GTP‐binding proteins with this organelle. Moreover, direct amino acid sequence analysis assigned at least three different small GTP‐binding proteins, ora3, o‐ral and o‐rab3, to the vesicular compartment. Furthermore, the data reflect the relative abundance of these three proteins on the vesicle membrane, thereby demonstrating the predominant occurrence of o‐rab3, the only exclusively synaptic vesicle specific small GTP‐binding protein.
Identification of phytanoyl‐CoA ligase as a distinct acyl‐CoA ligase in peroxisomes from cultured human skin fibroblasts
Authors: Pahan Kalipada , Cofer Joseph , Baliga Prabhakar , Singh Inderjit
Published: July 2002

Phytanic acid accumulates in excessive amounts in Refsum disease, a rare neurological disorder, due to a defect in its α‐oxidation enzyme system in peroxisomes. The activation of phytanic acid to phytanoyl‐CoA by phytanoyl‐CoA ligase is a prerequisite for its α‐oxidation. The studies described in this manuscript report that phytanoyl‐CoA ligase in peroxisomes is an enzyme distinct from the previously reported acyl‐CoA ligases.
High‐level expression of functional cGMP‐dependent protein kinase using the baculovirus system
Authors: Feil Robert , Müller Stefan , Hofmann Franz
Published: July 2002

The understanding of the structure and function of cGMP‐dependent protein kinase (cGMP kinase) has been hindered by the difficulty to obtain large quantities of functional enzyme. A recombinant baculovirus encoding bovine cGMP kinase la was constructed and purified. Infected insect cells synthesized large amounts of soluble and biologically active cGMP kinase Iα representing up to 10% of the total cell extract protein. The recombinant enzyme had an identical apparent molecular mass, cGMP affinity and kinase activity as the native bovine lung enzyme. The high‐level expression of functional cGMP kinase Iα should provide an excellent tool to study further the structure and function of cGMP kinase.
Changes in phospholipids during the cell cycle of myxomycete Physarum polycephalum
Authors: Makarov Peter R. , Rotaru Victor K. , Polteva Nina A. , Evtodienko Yuri V.
Published: July 2002

The rate of 32Pi incorporation into the main membrane phospholipid fractions, i.e. phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI), as well as their contents in the cells during synchronous growth of the myxomycete, Physarum polycephalum, have been studied. It has been found that both the phospholipid levels and the rates of 32Pi incorporation increase during the S phase till the early G2 phase, remain nearly constant during the G2 phase and fall to the initial level at the end of the G2 phase and in mitosis. It has been revealed that the rate of 32Pi incorporation into PC is very low compared to PE and PI, in spite of the fact that the PC level is the highest. The possible reasons of this phenomenon are discussed.
Molecular cloning of a bovine MSH receptor which is highly expressed in the testis
Authors: Vanetti Mirko , Schönrock Christiane , Meyerhof Wolfgang , Höllt Volker
Published: July 2002

A novel G protein‐coupled receptor (BDF3) was isolated from bovine genomic DNA by a combined approach of polymerase chain reaction (PCR) and hybridization techniques. The predicted amino acid sequence is 317 amino acids in length and displays 80% homology to the human α‐MSH receptor MC1. Stably transfected into CHO‐K1 cells, BDF3 mediates an increase of intracellular cAMP‐levels following incubation with NLE‐α‐MSH, a potent α‐MSH analog. The stimulation with ACTH1–10 is only moderate and γ‐MSH is ineffective. Northern blot analysis of bovine tissues revealed that the BDF3 gene is higly expressed in the testis as a single 2.3 kb mRNA species, suggesting an involvement of the BDF3 receptor in spermatogenesis.
Class I and IVa β‐tubulin isotypes expressed in adult mouse brain are glutamylated
Authors: Mary Jean , Redeker Virginie , Le Caer Jean-Pierre , Promé Jean-Claude , Rossier Jean
Published: July 2002

Several types of post‐translational modifications contribute to the high level of tubulin heterogeneity in the brain. An important modification is glutamylation of the major brain‐specific isotypes, such as class Ia/b of α‐tubulin and classes II and III of β‐tubulin. Here we describe experiments to determine if additional, minor tubulin isotypes, expressed in adult mouse brain, could also be glutamylated. Purified tubulin from adult mouse brain was cleaved with thermolysin. Proteolytically released carboxy‐terminal peptides of both α‐ and β‐tubulin were isolated by sequential anion exchange and reverse‐phase column‐chromatography. Anionic peptides were then characterized by amino acid sequencing and mass spectrometry. We show that brain‐specific class IVa and constitutive class I β‐tubulin isotypes can be glutamylated, at Glu434 and Glu441, respectively.