bluuebunny/crossref_metadata_2025 huggingface dataset:
Acute changes in myo‐inositol uptake and 22Na+ flux in murine neuroblastoma cells (N1E‐115) following insulin
Authors: Dunlop Marjorie , Dimitriadis Eva , Larkins Richard G.
Published: July 2002

myo‐Inositol uptake was investigated in a murine neuroblastoma clone (N1E‐115) to determine the effect of altered Na+,K+‐ATPase activity. The Na+ ionophone monensin, and veratridine, an alkaloid affecting voltage‐dependent Na+ entry, increased acute 22Na+ uptake and 22Na+ efflux from pre‐loaded cells, concomitant with enhanced myo‐inositol uptake. This effect was also seen following insulin. Insulin‐stimulated myo‐inositol uptake was inhibited by amiloride, ouabain and pyrithiamine. Amiloride inhibition suggests that activation of Na+/H+ exchange preceding Na+,K+‐ATPase activation is involved in insulin stimulation of myo‐inositol uptake. Pyrithiamine inhibition is an indication of prior activation of the Na+,K+‐ATPase α+ catalytic subunit by insulin. The results provide evidence that insulin contributes to the maintenance of Na+, K+‐ATPase in neuronal tissue.
Arachidonic acid‐induced release of calcium in permeabilized human neutrophils
Authors: Beaumier L. , Faucher N. , Naccache P.H.
Published: July 2002

The addition of arachidonic acid to a suspension of digitonin‐permeabilized human neutrophils was found to induce, in a dose‐dependent manner (ED50 about 15 μM), the release of calcium from internal stores. Arachidic acid was without effect, while linoleic acid and linolenic acid were (on a concentration basis) at least 5‐times less active than arachidonic acid. The activity of arachidonic acid appears to be due to the fatty acid itself and not to one of its metabolites. The pool of calcium mobilized by arachidonic acid includes that sensitive to inositol 1,4,5‐trisphosphate. These results demonstrate a significant intracellular role for arachidonic acid at the level of the internal mobilization of calcium in human neutrophils.
1H NMR and CD evidence of the folding of the isolated ribonuclease 50–61 fragment
Authors: Jiménez M.A. , Nieto J.L. , Herranz J. , Rico M. , Santoro J.
Published: July 2002

In our search for potential folding intermediates we have prepared and characterized the fragment of RNase A corresponding to residues 50–61. Proton chemical shift variations with temperature, addition of stabilizing (TFE) or denaturing agents (urea) provide a strong experimental basis for concluding that in aqueous solution this RNase fragment forms an α‐helix structure similar to that in the intact RNase A crystal. This conclusion lends strong support to the idea that elements of secondary structure (mainly α‐helices) can be formed in the absence of tertiary interactions and act as nucleation centers in the protein folding process.
Metabolism of plastid terpenoids: in vitro inhibition of phytoene synthesis by phenethyl pyrophosphate derivatives
Authors: Dogbo Odette , Bardat Françoise , Quennemet Joelle , Camara Bilal
Published: July 2002

The inhibition of partially purified phytoene synthetase activity from Capsicum annuum chromoplasts was investigated using aminophenethyl pyrophosphate and azidophenethyl pyrophosphate. These compounds were effective inhibitors of phytoene synthesis and kinetic analysis showed that they were competitive with respect to the substrate isopentenyl pyrophosphate. These data were strengthened by the ability of azidophenethyl pyrophosphate to photoinactivate irreversibly the activity of the enzyme complex. These results suggest that the primary targets of these analogs are at the level of isopentenyl pyrophosphate isomerase and geranylgeranyl pyrophosphate synthetase.
The amino‐terminal structure of oat phytochrome
Authors: Grimm Rudolf , Kellermann Josef , Schäfer Wolfram , Rüdiger Wolfhart
Published: July 2002

Phytochrome from etiolated oat seedlings was digested under carefully controlled conditions with trypsin. A dodecapeptide which contains the blocked amino‐terminus of phytochrome was isolated from the digestion mixture and analyzed. According to amino acid analysis and the results obtained in FAB mass spectrometry, the sequence of mature phytochrome starts with N‐acetylserine, corresponding to serine‐2 derived from the DNA sequence.
GTP‐binding proteins in human platelet membranes serving as the specific substrate of islet‐activating protein, pertussis toxin
Authors: Nagata Koh-ichi , Katada Toshiaki , Tohkin Masahiro , Itoh Hiroshi , Kaziro Yoshito , Ui Michio , Nozawa Yoshinori
Published: July 2002

Two GTP‐binding proteins serving as the specific substrate of islet‐activating protein (IAP), pertussis toxin, were purified from human platelet membranes as heterotrimers with an αβγ‐subunit structure. The α of the major IAP substrate had a molecular mass of 40 kDa and differed from that of Gi 1 or Go previously purified from brain membranes. The partial amino acid sequences of the 40 kDa α completely matched with the sequences which were deduced from the nucleotide sequences of the human Gi 2 α gene. On the other hand, the α of the minor IAP substrate purified from human platelets was about 41 kDa and cross‐reacted with an antibody raised against α of brain Gi 1 (Gi 1 α). These results indicate that the major IAP substrate present in human platelet membranes is a product of the Gi 2 α gene.
Small cytoplasmic RNA from mouse cells covalently linked to a protein
Authors: Vartapetian Andrey B. , Makarova Tatyana N. , Koonin Eugene V. , Agol Vadim I. , Bogdanov Alexey A.
Published: July 2002

A low‐molecular‐mass RNA from the cytoplasm of mouse Krebs II cells was found to be covalently linked to a protein with an apparent molecular mass of 13 kDa. The protein appears to be attached to the 5′‐terminus of the RNA molecule, which is approx. 20 nucleotides long.
Inhibition of insulin‐stimulated glucose transport in rat adipocytes by nucleoside transport inhibitors
Authors: Steinfelder H.J. , Joost H.G.
Published: July 2002

In isolated rat adipocytes, basal as well as insulin‐stimulated 3‐O‐methylglucose transport was inhibited nearly completely (maximal inhibition: 95%) by the nucleoside transport inhibitors dipyridamole (IC50 = 5 μM), nitrobenzylthioguanosine (20 μM), nitrobenzylthioinosine (35 μM) and papaverine (130 μM). Transport kinetics in the presence of 10 μM dipyridamole revealed a significant increase in the transport K
m value of 3‐O‐methylglucose (3.45±0.6 vs 2.36±0.29 mM in the controls) as well as a decrease in the Vmax value (4.84±0.95 vs 9.03±1.19 pmol/s per μl lipid in the controls). Half‐maximally inhibiting concentrations of dipyridamole were one order of magnitude higher than those inhibiting nucleoside (thymidine) uptake (0.48 μM). The inhibitory effect of dipyridamole (5 μM) reached its maximum within 30 s. The agent failed to affect insulin's half‐maximally stimulating concentration (0.075 nM) indicating that it did not interfere with the mechanism by which insulin stimulates glucose transport. Further, dipyridamole fully suppressed the glucose‐inhibitable cytochalasin B binding (IC50 = 1.65±0.05 μM). The data indicate that nucleoside transport inhibitors reduce glucose transport by a direct interaction with the transporter or a closely related protein. It is suggested that glucose and nucleoside transporters share structural, and possibly functional, features.
Influence of thyroid status on the membranes of rat liver mitochondria Unique localization of L‐glycerol‐3‐phosphate dehydrogenase
Authors: Beleznai Z. , Amler E. , Rauchová H. , Drahota Z. , Jancsik V.
Published: July 2002

The effect of thyroid status on the physical properties of rat liver mitochondrial membranes and on the lipid microenvironment of proteins was investigated. The steady‐state fluorescence anisotropy of diphenyl‐1,3,5‐triene and 1‐[4‐{trimethylaminophenyl} phenyl]‐6‐phenylhexa‐1,3,5‐triene revealed an increase of the order of the membranes with the increase of hormone level. Protein arrangement in the inner mitochondrial membrane altered with the thyroid status, which was reflected by digitonin subfractionation of mitochondria. The microenvironment of FAD‐linked L‐glycerol‐3‐phosphate dehydrogenase was dramatically influenced by thyroxine.
A new monoclonal antibody recognizing the amino‐terminal consensus sequence of vertebrate intermediate filament proteins
Authors: Escurat Michel , Phamgia Hai , Huc Claude , Pouplard-Barthelaix Annick , Boitard Christian , Bach Jean-François , Gros François , Portier Marie-Madeleine
Published: July 2002

The mouse monoclonal antibody ME 101 raised against human peripherin, an intermediate filament protein (IFP) specific to well defined neuronal populations, recognizes all the major classes of vertebrate IFP in immunoblotting assays. Desmin, GFAP, vimentin, peripherin and the lightest neurofilament protein (NF‐L) were cleaved into carboxy‐ and amino‐terminal halves by N‐chlorosuccinimide at their unique trytophan residue. Whereas the antibody directed against the epitope common to every IFP (intermediate filament antigen or IFA) and located on the carboxy‐terminal end of the rod domain recognizes the carboxy‐terminal half, the ME 101 antibody, as the present study illustrates, recognizes specifically the amino‐terminal half. From the amino acid sequence data of IFP, it is deduced that the cognate epitope is localized on the amino‐terminal part of coil la.
Extracellular Ca2+‐activated K channel in coronary artery smooth muscle cells and its role in vasodilation
Authors: Inoue Isao , Nakaya Yutaka , Nakaya Shuichiro , Mori Hiroyoshi
Published: July 2002

An extracellular Ca2+‐activated K channel (KR channel), having a conductance of 30 pS, was identified in isolated single smooth muscle cells from porcine coronary artery. The KR channel was active at > 10−5 M Ca2+, and was blocked by 4‐aminopyridine (4AP). At < 10−6 M Ca2+, the KR channel became inactive, but could be activated by 2‐nicotinamidethyl nitrate (nicorandil), or by 4‐acetamide‐4′‐isothiocyanostilbene‐2,2′‐disulfonic acid (SITS) applied to the pipette solution. It was found that there is a close correlation between the KR channel activity and cell contraction: cells contracted under conditions in which the KR channel became inactive, but were relaxed when the KR channel was active. As the KR channel is highly active in cells in physiological saline, we suggest that it controls the tonus of the coronary artery, as an endogenous dilating factor.
Genetic transformation of mouse cultured cells with the help of high‐velocity mechanical DNA injection
Authors: Zelenin Alexander V. , Titomirov Alexander V. , Kolesnikov Viktor A.
Published: July 2002

NIH 3T3 mouse cells were transfected by the plasmid pSV3neo (G418‐resistant) with the help of high‐velocity mechanical DNA injection based on the principle of bombarding cells with tungsten particles covered with the DNA. Stable transformants were obtained. Dot‐hybridization and Southern analysis revealed the integration into the genome of 5–20 copies per cell of original plasmid DNA. The plasmid DNA was shown to have tandem organization.
Characterization of platelet‐activating factor‐induced elevation of cytosolic free calcium concentration in eosinophils
Authors: Kroegel Claus , Pleass Robert , Yukawa Tatsuo , Chung K.Fan , Westwick John , Barnes Peter J.
Published: July 2002

In order to evaluate the role of calcium in the activation processes in eosinophils induced by platelet‐activating factor (PAF), we investigated the changes in free cytoplasmatic Ca2+ concentration using fura‐2. PAF causes a rapid and transitory rise of the intracellular free calcium ion concentration ([Ca2+]i) in purified guinea pig eosinophils of approx. 1000 nM above a basal level of 120.7 ± 36.5 nM (n = 10). The effect was dose‐related with a maximum rise at 1000 nM PAF and an EC50 of 17.4 nM and specifically inhibited by the PAF antagonist WEB 2086 with an IC50, of 95.5 nM. WEB 2086 did not affect either the leukotriene B4‐ or the fMet‐Leu‐Phe‐induced elevation of [C2+]i. The response to PAF was dependent on external Ca2+ as it was significantly inhibited by EGTA (85.6 ± 5.4%) and Ni2+ (95.8 ± 2.1 %) but not by the dihydropyridine antagonist nimodipine. We conclude that Ca2+ entry via receptor‐operated Ca2+ channels may be involved in PAF‐induced degranulation of eosinophils.
Neuroactive metabolises of L‐tryptophan, serotonin and quinolinic acid, in striatal extracellular fluid effect of tryptophan loading
Authors: During Matthew J. , Freese Andrew , Heyes Melvyn P. , Swartz Kenton J. , Markey Stanford P. , Roth Robert H. , Martin Joseph B.
Published: July 2002

Extracellular fluid levels of the neurotoxin quinolinic acid in the corpus striatum of rats, measured by in vivo microdialysis, were increased in a dose‐dependent manner following the intraperitoneal administration of tryptophan. The lowest dose of tryptophan (12.5 mg/kg), equivalent to about 5% of the normal daily intake, increased peak quinolinic acid levels nearly 3‐fold. At higher doses of tryptophan (up to 250 mg/kg), concentrations of quinolinic acid increased over 200‐fold and exceeded potentially neurotoxic levels (10 μM). In contrast, the increase in extracellular serotonin following even the highest tryptophan dose was small (less than 2‐fold). These data indicate that quinolinic acid is present in them extracellular fluid where it may function as a neuromodulator and that it is very responsive to physiological changes in precursor availability.
Photoaffinity labeling of the mammalian dopamine transporter
Authors: Sallee Floyd R. , Fogel Evan L. , Schwartz Eric , Choi Sung-Mo , Curran Dennis P. , Niznik Hyman B.
Published: July 2002

Dopamine uptake; Transporter; Photoaffinity labeling; Deglycosylation
In vitro interaction of mutagenic chromium(VI) with red blood cells
Authors: Branca M. , Dessi A. , Kozlowski H. , Micera G. , Serra M.V.
Published: July 2002

Glutathione; Chromium; ESR; (Red blood cell)
Chlorophyll a and β‐carotene content in the D1/D2/cytochrome b‐559 reaction center complex from spinach
Authors: Kobayashi Masami , Maeda Hiroyuki , Watanabe Tadashi , Nakane Hiroyuki , Satoh Kimiyuki
Published: July 2002

Pigment stoichiometries in the D1/D2/cytochrome b‐559 reaction center complex from spinach have been studied by spectrophotometry and normalphase silica HPLC of the solvent extracts. Based on the well‐accepted molar ratio Pheo a/P680 = 2, the results are summarized by Chl a/P680 = 6.01 ± 0.37 (n = 24) and β‐carotene/P680= 1.84 ± 0.11 (n = 4). These stoichiometries are significantly different from those of bacterial reaction center complexes.
Neuraxin corresponds to a C‐terminal fragment of microtubule‐associated protein 5 (MAP5)
Authors: Kirsch Joachim , Littauer Uriel Z. , Schmitt Bertram , Prior Peter , Thomas Leo , Betz Heinrich
Published: July 2002

From cloned DNA, neuraxin has been identified as a tubulin binding protein of predicted molecular weight of 94 kDa. The deduced sequence of the rat protein exhibits high homology to the C‐terminal region of mouse microtubule‐associated protein 5 (MAP5). Here, we show that different neuraxin antibodies recognize MAP5, but fail to detect a protein of 94 kDa, in subcellular and microtubular fractions of the rat central nervous system. Furthermore, tubulin binding by neuraxin was found to be dependent on taxol. These data are consistent with neuraxin corresponding to a C‐terminal fragment of MAP5 that contains a low‐affinity tubulin binding site.
The cell adhesion molecule Cell‐CAM 105 is an ecto‐ATPase and a member of the immunoglobulin superfamily
Authors: Aurivillius Magnus , Hansen Ole C. , Lazrek Mohamed B.S. , Bock Elisabeth , Öbrink Björn
Published: July 2002

Cell‐CAM 105 (C‐CAM), a cell adhesion molecule in rat hepatocytes, was digested with trypsin, and peptides were isolated and sequenced by Edman degradation. The sequences of 4 peptides agreed with different regions of rat liver ecto‐ATPase. Detailed biochemical analyses confirmed the identity between C‐CAM and the ecto‐ATPase. C‐CAM/ecto‐ATPase is a transmembrane protein having 4 immunoglobulin‐like domains in the extracellular portion, demonstrating membership of the immunoglobulin superfamily. The ATPase activity suggests that ATP might influence cell adhesion, which would explain the inhibitory effect of exogenously added ATP on adhesion of several cell types.
Nerve growth factor stimulates protein kinase C translocation in PC12 cells
Authors: Kondratyev A.D. , Popova O.N. , Severin S.E. , Choladze M.A. , Shmyrev I.I. , Tubasheva I.A. , Zotova E.E. , Posypanova G.A. , Severin E.S.
Published: July 2002

The effect of nerve growth factor (NGF) on the activity and intracellular localization of protein kinase C (PKC) in pheochromocytoma PC12 cells was studied. By using immunoblotting, immunofluorescence method and phorbol ester binding, NGF was found to induce PKC translocation from the cytoplasm into the cell membrane. This process was accompanied by an increase in the protein kinase activity in the membrane fraction. Translocation was completely blocked by H‐7, a protein kinase inhibitor potentiating the neurite‐stimulating activity of NGF.
cDNA cloning and sequencing of component C5 of proteasomes from rat hepatoma cells
Authors: Tamura Tomohiro , Tanaka Keiji , Kumatori Atsushi , Yamada Fumi , Tsurumi Chizuko , Fujiwara Tsutomu , Ichihara Akira , Tokunaga Fuminori , Aruga Rie , Iwanaga Sadaaki
Published: July 2002

Proteasomes are multicatalytic proteinase complexes consisting of a set of non‐identical polypeptide subunits. A cDNA for component C5 of rat proteasomes was isolated by screening a Reuber H4TG hepatoma cell cDNA library using synthetic oligodeoxynucleotide probes corresponding to partial amino acid sequences of the protein. The polypeptide deduced from the open reading frame consisted of 240 amino acid residues with a calculated molecular weight of 26479. Computer analysis revealed little similarity of C5 to other proteins reported so far. The primary structure of C5 showed partial sequence homology to that of another component C3, but no regions of homology with the sequence of component C2. Thus C5 is concluded to be a new type of subunit of the proteasome complex.
Rats with physically disconnected hypothalamo‐pituitary tracts no longer contain vasopressin‐oxytocin gene transcripts in the posterior pituitary lobe
Authors: Mohr Evita , Zhou An , Thorn Niels A. , Richter Dietmar
Published: July 2002

In rats, vasopressin‐ and oxytocin‐encoding mRNAs are present in the posterior but absent in the anterior lobe of the pituitary gland. RNase protection experiments indicate that in the posterior pituitary and hypothalamus identical transcriptional start points are used. Furthermore, the two transcripts from posterior pituitary and hypothalamus show identical nucleotide sequences. Animals operated by paired electrical lesions in such a way that connections between the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus and the posterior pituitary lobeare destroyed continue to express the vasopressin and oxytocin gene in the hypothalamus but not in the posterior pituitary. Operated animals subjected to chronic intermittent salt loading for 6 days similarly contain vasopressin and oxytocin encoding transcripts in the hypothalamus but not in the posterior pituitary.
Expression of catalytically active radish 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase in Escherichia coli
Authors: Ferrer Albert , Aparicio Cristina , Nogués Núria , Wettstein Annette , Bach Thomas J. , Boronat Albert
Published: July 2002

Two fragments of a cDNA encoding radish 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMGR) were cloned into the vector pET‐8c and expressed in Escherichia coli. The large fragment, encoding both the membrane and the cytosolic domains, was expressed at low level, essentially as an insoluble protein without enzymatic activity. In contrast, the fragment encoding only the cytosolic domain was expressed at a high level in a catalytically active form. The amount of soluble active enzyme in cell‐free extracts of E. coli dramatically increased when the temperature during the induction was lowered from 37°C to 22°C.
Extreme variations in the ratios of non‐synonymous to synonymous nucleotide substitution rates in signal peptide evolution
Authors: Garcia-Maroto Federico , Castagnaro Atilio , de la Hoz Pilar Sanchez , Maraña Carmen , Carbonero Pilar , García-Olmedo Francisco
Published: July 2002

Nucleotide sequences encoding signal peptidcs from the precursors of α‐amylase/trypsin inhibitors from cereals are homologous to those corresponding to the precursors of thaumatin II and of plastocyanins. Non‐synonymous (K
A) and synonymous (K
S) rates of nucleotide substitutions have been calculated for all possible binary combinations. Extreme variation in K
A/K
S ratios has been observed, from the 0.167 average found within the plastocyanin family to an average of 1.90 calculated for the inhibitors/thaumatin II transition. A similar calculation has been carried out for the signal peptide sequences of thionins. which are unrelated to those of the α‐amylase trypsin inhibitor family, and an average K
A/K
S of 0.12 has been obtained. This variation can be largely explained in terms of an empirical index of stability related to amino acid composition and seems to be independent of functional constraints.
Effects of the general anaesthetic Propofol on the Ca2+‐induced permeabilization of rat liver mitochondria
Authors: Eriksson Ove
Published: July 2002

The molecular mechanisms of the Ca2+‐induced permeabilization of rat liver mitochondria was evaluated by studying a new effect of the commonly used general anaesthetic Profol(2,6‐diisopropylphenol). The compound was found to induce an apparent uptake of Ca2+ at steady‐state in the Ca2+‐distributed between the medium and the mitochondria, and to inhibit swelling and release of accumulated Ca2+ induced by inorganic phosphate, t‐butyl hydroperoxide, diamide or FCCP plus Ruthenium red. The compound did not stimulate the activity of the Ca2+‐uniporter and it is concluded that the effects seen are due to the inhibition of the Ca2+‐dependent, unspecific permeability increase. The results suggest two mechanisms whereby Propofol stabilizes the mitochondrial membrane in the presence of Ca2+: (i) by interaction with the putative pore, thus causing its closure; and (ii) by scavenging of free radicals thus inhibiting its opening during oxidative stress.
Antibodies against synthetic amphipathic helical sequences of surfactant protein SP‐B detect a conformational change in the native protein
Authors: Fan Bunchen R. , Bruni Roberta , Taeusch H.William , Findlay Richard , Waring Alan J.
Published: July 2002

Synthetic peptides based on the native human sequence of surfactant protein B have been used to generate polyclonal monospecific antibodies against specific segments of the native SP‐B protein. Circular dichroism analysis of the synthetic peptides shows they have a dominant helical content in structure promoting environments and tensiometric measurements indicate these peptides lower surface tension at air—water interfaces implying that they contain amphipathic alpha helical motifs. Antibodies directed against the C‐terminal segment of SP‐B react with the native protein in the oxidized and reduced state. Antibodies directed against the N‐terminal sequence of SP‐B react with the native protein only in the reduced state suggesting that this domain has a conformation dependent on disulfide bond formation.
Antigen receptor‐mediated calcium signals in B cells as revealed by confocal fluorescence microscopy
Authors: Yamada Hiroshi , Mizuguchi Junichiro , Nakanishi Mamoru
Published: July 2002

A confocal fluorescence microscope was used to study the antigen receptor‐mediated calcium signals in B cells. Anti‐IgD binding to B lymphoma cells (BAL17) increased the intracellular calcium concentration with short lag times. Confocal fluorescence images of the fluo‐3‐loaded BAL17 cells showed that the intracellular calcium ion concentrations increased non‐homogeneously, suggesting that the calcium signals transferred not only to the cytoplasm but also to the nucleus.
An impaired peroxisomal targeting sequence leading to an unusual bicompartmental distribution of cytosolic epoxide hydrolase
Authors: Arand Michael , Knehr Michael , Thomas Helmut , Zeller Hans-Dieter , Oesch Franz
Published: July 2002

To gain an understanding of the mechanism by which the subcellular distribution of cytosolic epoxide hydrolase (cEH) is directed, we have analyzed the carboxy terminal region of rat liver cEH by means of cDNA cloning to define the structure of its possible peroxisomal targeting sequence (PTS). Purified cEH was subjected to peptide analysis following endoproteinase Glu‐C digestion and HPLC‐separation of the fragments. The obtained sequence information was used to perform PCR experiments resulting in the isolation of a 680 bp cDNA clone encoding the carboxy terminus of cEH. The deduced amino acid sequence displays a terminal tripeptide Ser‐Lys‐Ile which is highly homologous to the PTS (Ser‐Lys‐Leu) found in other peroxisomal enzymes. This slight difference appears to be sufficient to convert the signal sequence into an impaired and therefore ambivalent PTS, directing the enzyme partly to the peroxisomes and allowing part to reside in the cytosol.
Prokaryotic calcium‐binding protein of the calmodulin superfamily Calcium binding to a Saccharopolyspora erythraea 20 kDa protein
Authors: Bylsma Niels , Drakenberg Torbjörn , Andersson Ingrid , Leadlay Peter F. , Forsén Sture
Published: July 2002

The EF‐hand calcium‐binding protein from Saccharopolyspora erythraea has been shown, using 113Cd NMR, to possess three Cd2+‐ion binding sites. This indicates that of the four EF‐hand motifs in the molecule, one (probably site 2) is unable to bind Cd2+‐ions. Data from the titration of the protein with Ca2+, in the presence of Quin2, were fitted to a curve calculated on the assumption that the protein contains three high affinity Ca2+ binding sites, two of which (pK
1 = 8.0. pK
2 = 9.0) are strongly cooperative, and one single site (pK
3 = 7.5). Preliminary 1H NMR experiments indicate marked structural changes upon Ca2+‐binding.
In vivo import of yeast adenylate kinase into mitochondria affected by site‐directed mutagenesis
Authors: Magdolen Viktor , Schricker Roland , Strobel Gertrud , Germaier Herbert , Bandlow Wolfhard
Published: July 2002

Site‐directed mutagenesis and deletions were used to study mitochondrial import or a major yeast adenylate kinase, Aky2p. This enzyme lacks a cleavable presequence and occurs in active and apparently unprocessed form both in mitochondria and cytoplasm. Mutations were applied to regions known to be surface‐exposed and to diverge between short and long isoforms. In vertebrates, short adenylate kinase isozymes occur exclusively in the cytoplasm, whereas long versions of the enzyme have mitochondrial locations. Mutations in the extra loop of the yeast (long‐form) enzyme did not affect mitochondrial import of the protein, whereas variants altered in the central, N‐ or C‐terminal parts frequently displayed increased or, in the case of a deletion of the 8 N‐teminal triplets, decreased import efficiencies. Although the N‐terminus is important for targeting adenylate kinase to mitochondria, other parameters like internal sequence determinants and folding velocity of the nascent protein may also play a role.
Messenger RNA sorting in enterocytes Co‐localization with encoded proteins
Authors: Rings Edmond H.H.M. , Büller Hans A. , de Boer Piet A.J. , Grand Richard J. , Montgomery Robert K. , Lamers Wouter H. , Charles Robert , Moorman Antoon F.M.
Published: July 2002

This study describes the intracellular compartmentalization of three different mRNAs in the polarized rat fetal enterocyte. They encode proteins that are known to be localized within different regions of the epithelial cell namely (i) the apical, membrane‐bound glycoprotein, lactase‐phlorizin hydrolase (lactase), (ii) the mitochondrially localized enzyme, carbamoylphosphate synthetase (CPS), and (iii) the cytoplasmically localized enzyme, phosphoenolpyruvate carboxykinase (PEPCK). These mRNAs are found in close proximity to their respective protein products, i.e. the apical membrane, mitochondria and cytoplasm, respectively. The significance or these observations is twofold: (i) they indicate that mRNAs are sorted into specific domains of the cytosol of intestinal epithelial cells: and (ii) they imply the presence or two distinct pathways of mRNA targeting one that allows transport of mRNAs that are translated on ribosomes associated with the rough endoplasmic reticulum (lactase mRNA), and the other that allows sorting of mRNAs that are translated on free polysames (CPS and PEPCK mRNA).
Effects of okadaic acid on the activities of two distinct phosphatidate phosphohydrolases in rat hepatocytes
Authors: Gomez-Muñoz Antonio , Hatch Grant M. , Martin Ashley , Jamal Zahirali , Vance Dennis E. , Brindley David N.
Published: July 2002

Incubation of hepatocytes with okadaic acid displaced the N‐ethylmaleimide‐sensitive phosphatidate phosphohydrolase from the membrane fraction into the cytosol and partially prevented the oleate‐induced movement of phosphohydrolase from cytosol to membranes. However, higher concentrations of oleate still caused translocation and activation of the phosphohydrolase. This enzyme is stimulated by Mg2+, and is probably involved in glycerolipid synthesis. Okadaic acid also decreased the concentration of diacylglycerol within the hepatocytes. Okadaic acid had no observable effect on the activity of an N‐ethylmaleimide‐insensitive phosphatidate phosphohydrolase which remained firmly attached to membranes. This activity is not stimulated by Mg2+ and is probably involved in signal transduction by the phospholipase D pathway.
Thermoluminescence evidence for light‐induced oxidation of tyrosine and histidine residues in manganese‐depleted photosystem II particles
Authors: Allakhverdiev Suleyman I. , Klimov Vyacheslav V. , Demeter Sandor
Published: July 2002

In the thermoluminescence (TL) glow curve of photosystem II, particles depleted of manganese, a tyrosine modifier, 7‐chloro‐4‐nitrobenz‐2‐oxa‐1,3‐diazole (NBD) a bolishes the TL band appearing around −55°C (TL−55). Addition of a histidine modifier, diethylpyrocarbonate results in the disappearance of the band peaking around −30°C (TL−30). NBD treatment also abolishes the EPR signal IIfast of oxidized tyrosine donor, Y21 and inhibits the electron transport from diphenylcarbazide to 2,6‐dichlorophenol‐indophenol. It is concluded that the TL−55 and TL−30 bands can be assigned to oxidized tyrosine (Y2
+) and histidine (His+) residues, respectively, which participate in electron transfer from manganese to the reaction center of chlorophyll, P680+.
Organic solvent changes the chymotrypsin specificity with respect to nucleophiles
Authors: Gololobov Mikhail Yu. , Voyushina Tatyana L. , Stepanov Valentin M. , Adlercreutz Patrick
Published: July 2002

In α‐chymotrypsin‐catalyzed acyl‐transfer reactions in water the specificity of the enzyme (the nucleophile reactivity of amino acid amides) is correlated with the substrate hydrophobicity and increases as the hydrophobicity of the side chain of the amino acid amides is increased. In a low water system (4% H2O) bulky amino acid amides are less efficient nucleophiles. The specificity of α‐chymotrypsin towards the amino acid amides in acyl transfer reactions in this case does not depend on the hydrophobicity of the amino acid side chains but correlates with their size. Therefore, different factors can be responsible for the specificity of enzymes in water and in a mainly organic medium.
The 5′ untranslated region of the human cellular glutathione peroxidase gene is indispensable for its expression in COS‐7 cells
Authors: Kurata Hiroshi , Kamoshita Keiichi , Kawai Eiko , Sukenaga Yoshikazu , Mizutani Takaharu
Published: July 2002

We studied the expression of the human cellular glutathione peroxidase (GPx) gene, from which a key enzyme containing selenocysteine (Scy) at the active site is produced. Expression of some human GPx gene mutants in COS‐7 cells revealed that the 5′ untranslated region (utr) was necessary for expression of the GPx gene, since mutant genes having 10 base pairs (bps) at the 5′utr (the complete had 311 bps) expressed GPx at very low levels. The genes with 311 or 408 bps at the 5′utr were better expressed than those having 257 bps. The GPx gene having 133 bps at the 3′utr (80 bps shorter than the entire length) was highly expressed. This deletion did not influence expression. We constructed some mutants in which 3 bases were altered at the upstream region of the Scy UGA codon in the frame of the GPx gene, by site‐directed mutagenesis. GPx expression decreased but the expression was restored. Therefore, the upstream region of the in‐frame Scy codon was not essential in the Scy decoding mechanisms. Finally, the 5′utr was essential for the expression of GPx gene. However, the deletion of a part of the 3′utr and the site‐directed mutation upstream of the Scy codon did not show drastic effects on the expression.
Nucleotide sequence of the genes for ribosomal proteins HS15 and HSH from Haloarcula marismortui: an archaeon‐specific gene cluster
Authors: Arndt Evelyn , Steffens Christina
Published: July 2002

The nucleotide sequences of the genes for two ribosomal proteins, H515 and HSH, from the archaeon Haloarcula marismortui, have been determined. The genes were found in a cluster together with another open reading frame with a probable regulatory function. HS15 S and HSH have counterparts in eucarya. HS15 is significantly homologous to S19 from frog (Xenopus laevis). HSH is related to S37 from yeast (Saccharomyces cerevisiae) and S27 from fly (Drosophila melanogasier), as well as to other members of the S27 family. Eubacterial counterparts were not found, suggesting, that these proteins are ‘extra proteins’ that are absent in eubacterial ribosomes.
Augmentation of retinoic acid‐induced granulocytic differentiation in HL‐60 leukemia cells by serine/threonine protein phosphatase inhibitors
Authors: Morita Koichi , Nishikawa Masakatsu , Kobayashi Kazuhiko , Deguchi Katsumi , Itoh Masaaki , Nakano Takeshi , Shima Hiroshi , Nagao Minako , Kuno Takayoshi , Tanaka Chikako , Shirakawa Shigeru
Published: July 2002

To evaluate the involvement of protein phosphatases (PP) in differentiation of human myclogenous leukemia HL‐60 cells, we made use of potent inhibitors of PP1 and PP2A, calyculin‐A (CAL‐A) and okadaic acid (OKA). CAL‐A and OKA could augment all‐trans retinoic acid (ATRA)‐induced granulocytic differentiation, whereas the differentiation toward macrophage lineage by 12‐o‐tetradecanoylphorbol acetate (TPA) was unchanged in the presence of CAL‐A. CAL‐A augmented the phosphorylation or 18K, 23K and 30K proteins induced by ATRA. The PP1 and PP2A were identified and were present mainly in the cytosol of HL‐60 cells. These results suggest that either PP1 or PP2A or both may be involved in regulating granulocytic differentiation or HL‐60 cells.
PsaL subunit is required for the formation of photosystem I trimers in the cyanobacterium Synechocystis sp. PCC 6803
Authors: Chitnis Vaishali P. , Chitnis Parag R.
Published: July 2002

When membranes of the wild type strain of the cyanobacterium Synechocystis sp. PCC 6803 were solubilized with detergents and fractionated by sucrose‐gradient ultracentrifugation, photosystem I could be obtained as trimers and monomers. We could not obtain trimers from the membranes of any mutant strain that lacked PsaL subunit. In contrast, absence of PsaE, PsaD, PsaF, or PsaJ did not completely abolish the ability of photosystem I to form trimers. Furthermore, PsaL is accessible to digestion by thermolysin in the monomers but not in the trimers of photosystem I purified from wild type membranes. Therefore, PsaL is necessary for trimerization of photosystem I and may constitute the trimer‐forming domain in the structure of photosystem I.
Properties of α1‐antitrypsin secreted by human adenocarcinoma cell lines
Authors: Kataoka Hiroaki , Seguchi Kohji , Inoue Teruhiko , Koono Masashi
Published: July 2002

α1‐Antitrypsin; (α1‐AT) produced by various human carcinoma (non‐hepatoma) cell lines were analyzed. Five out of eight cell lines secreted detectable amounts of α1‐AT into the conditioned media. All were adenocarcinoma cell lines. The tumor cell‐derived α1‐ATs had higher molecular weights (MW) than the normal plasma form. Most of this difference was an overall reflection of altered N‐glycosylation. As judged by binding of lectins, the glycosylation had shifted towards higher levels of triantennary oligosaccharides and higher levels of fucosylation. The conditioned media also contained lower MW α1‐AT species, possibly, proteolytically cleaved forms.
Model peptides to study the effects of P2 and P3 substitutions in statine‐containing HIV proteinase inhibitors
Authors: Hui Kwan Y. , Hermann Robert B. , Manetta Joseph V. , Gygi Theresa , Angleton Eddie L.
Published: July 2002

Through a series of synthetic model peptides, we have examined the structural requirements of the P2 and P3 residues in statine‐based HIV protease (PR) inhibitors. Results agree with the general observations that, the more bulky the P3 aromatic hydrophobic side chain, the more potent is the inhibitor. At P2, an isopropyl side chain is critical in maintaining potency. Three‐dimensional modeling demonstrates that the steric bulk of a leucyl residue or the unfavorable energy transfer, from water to enzyme, for a basic amino acid residue at P2 markedly compromises activity. A naphthylalaninyl‐valyl P3‐P2 substituted analogue inhibits PR with an IC50 value of 6 nM, and was also effective as an antiviral agent.
Characterization of metabotropic glutamate receptors coupled to a pertussis toxin sensitive G‐protein in bovine brain coated vesicles
Authors: Martín Mairena , Sanz Juana M. , Cubero Ana
Published: July 2002

Glutamate metabotropic receptors (mGluRs) in bovine brain coated vesicles have been characterized by pharmacological and kinetic binding experiments. Saturation experiments revealed a single binding site with a K
d = 607.9 ± 78.5 nM and a B
max = 6.45 ± 0.88 pmol/mg protein. The specific binding of l‐[3H]glutamate to mGluRs is regulated by guanine nucleotides. Guanosine‐5'‐triphosphate (GTP; 100 μM) shifts the agonist competition curves to the right, increasing the IC50 values. Pertussis toxin treatment produces a pharmacological binding profile for quisqualate similar to that obtained in the presence of 100μM GTP. These results indicate the presence of metabotropic glutamate receptors in coated vesicles and its coupling to a pertussis toxin sensitive G‐protein.
Conformational changes of the α1‐proteinase inhibitor affecting its cholesterol binding ability
Authors: Janciauskiene Sabina , Eriksson Sten
Published: July 2002

The effect of conformational changes of the α1‐proteinase inhibitor (α1pI) on α1PI‐cholesterol complex (1:2 mol/mol) formation in vitro was studied with electrophoretic and gel Chromatographic methods. Native α1PI was modified by adding free thiol agents such as glutathione, cysteine HCl, or dl‐homocysteine, by heating, or by cleavage with pancreatic elastase or trypsin. Conformational changes of the α1PI molecule induced by these procedures were all accompanied by a loss of its ability to bind cholesterol in vitro under standard experimental conditions. The data suggest α1PI‐cholesterol binding to be affected by both direct and indirect modifications of the α1PI‐reactive center, that is situated on a mobile peptide loop.
Down‐regulation of ubiquitin gene expression during differentiation of human leukemia cells
Authors: Shimbara Naoki , Sato Chiharu , Takashina Makoto , Tanaka Tatsuo , Tanaka Keiji , Ichihara Akira
Published: July 2002

Ubiquitin, which is ligated covalently to target proteins for their acquisition of a variety of functions, is encoded by multiple unique genes in human cells: two distinct poly‐ubiquitin genes with tandemly repeated sequences of 3 or 9 moieties and two mono‐ubiquitin genes fused with small and large ribosomal proteins. We found that all classes of ubiquitin genes as well as the two genes encoding the ribosomal proteins S17 and L31 were expressed at abnormally high levels in various hematopoietic malignant tumor cells. In contrast, in vitro terminal differentiation of various immature leukemic cell lines, such as HL‐60 promyelocytic leukemia cells and K562 erythroleukemia cells into monocytic, granulocytic and erythroid cells, induced by various agents was found to cause rapid and marked down‐regulation of ubiquitin expression, irrespective of the cell type, direction of differentiation or type of signal. These findings suggest that the expressions of the multiple ubiquitin genes, coordinated with those of the ribosomal protein genes, are in a dynamic state during growth and differentiation of leukemia cells.
Coexpression of type I and type II IL‐1 receptors in the murine T helper 2 cell line D10N
Authors: Brigelius-Flohé R. , McCarthy J.E.G. , Resch K. , Szamel M. , Martin M.
Published: July 2002

IL‐1 receptor heterogeneity in murine lymphocytes was investigated by cross‐linking to [125I]IL‐1α and competition with IL‐1 receptor antagonist, and the molecular identity of the IL‐1 receptors was identified with PCR using primers specific for type I and type II IL‐1 receptors. The thymoma cell line EI4 6.1 exhibits exclusively the 80 kDa receptors which proved to be the type I receptor according to PCR analysis. In the pre‐B cell line 70Z/3, predominantly a 60 kDa type II receptor but also a trace of type I receptor can be identified by PCR. The Th2 cell line D10N expresses both types of IL‐1 receptors in equivalent amounts according to cross‐linking experiments and PCR. The proliferative response of D10N cells to IL‐1 is inhibited by IL‐1 ra which according to cross‐linking affects the binding to the type I receptor only. It is concluded that coexpression of both types of IL‐1 receptors might be a characteristic of murine Th2 cells and that their growth‐dependence on IL‐1 is mediated by the type I receptor.
Expression and functional characterisation of a synthetic version of the human D4 dopamine receptor in a stable human cell line
Authors: McHale Mark , Coldwell Martin C. , Herrity Nicole , Boyfield Izzy , Winn Fiona M. , Ball Stuart , Cook Ted , Robinson Jeff H. , Gloger Israel S.
Published: July 2002

A synthetic version of the human D4 (hD4) dopamine receptor was prepared. The G/C content of the natural gene was reduced by 14% without altering the amino acid composition of the corresponding protein sequence. HEK293 cells were transfected with the synthetic hD4 gene and stable clones resistant to G418 selected. The hD4 receptor expressed from the synthetic gene had identical pharmacological characteristics to the native hD4 receptor [(1991) Nature 350, 610–619; (1992) Nature 358, 149–152]. Functional studies with cells expressing the synthetic hD4 gene indicated negative coupling of this receptor to adenylate cyclase.
Isolation of a potassium‐selective ion channel from the plasma membrane of the broad bean Vicia faba L
Authors: Zeilinger C.
Published: July 2002

Potassium ions are essential for plant nutrition and play an important role in osmoregulation and plant movement. Recent patch clamp investigations and molecular analysis have identified voltage‐dependent potassium channels responsible for translocation of this ion across plant membranes. In order to characterize these transporters on the biochemical level, a potassium channel protein was isolated from Vicia faba under preservation of its functional integrity. Potassium channel activity was monitored by tracer flux and black lipid bilayer measurements. The capability for K+ transport copurified with a 67 kDa protein from V. faba plasma membranes. A monoclonal antibody directed against the 67 kDa protein blocked channel activity. Single channels with a unit conductance of 40 pS were highly selective for K+ over Na+.
Structure of membrane‐bound human factor Va
Authors: Stoylova Svetla , Mann Kenneth G. , Brisson Alain
Published: July 2002

Coagulation factor Va is an essential cofactor which combines with the serine protease factor Xa on a phospholipid surface to form the prothrombinase complex. In the present study, the structure of factor Va interacting with lipid surfaces containing phosphatidylserine was studied by electron microscopy. Two‐dimensional crystals of factor Va were obtained on planar lipid films under quasi‐physiological conditions. The two‐dimensional projected structure of factor Va was calculated at a resolution of 2 nm, revealing dimers of factor Va arranged on the surface lattice with the symmetry of the plane group p2. Average unit cell dimensions are a = 14.4 nm, b = 8.8 nm, γ = 107°. Each factor Va molecule presents two distinct domains of protein density consisting of one small domain, of 3 nm in diameter, connected to a larger domain of about 6 nm × 4.5 nm. The projected structure of factor Va covers an area equivalent to about fifty phospholipid molecules. In addition, edge‐on views of factor Va molecules bound to liposomes reveal a globular structure connected through a thin stem to the liposome surface. A three‐dimensional model of membrane‐bound factor Va is proposed.
Susceptibility of tenascin to degradation by matrix metalloproteinases and serine proteinases
Authors: Imai Kazushi , Kusakabe Moriaki , Sakakura Teruyo , Nakanishi Isao , Okada Yasunori
Published: July 2002

The degradation of tenascin purified from human melanoma cells was examined by treatment with matrix metalloproteinases (MMPs) and serine proteinases. Among eight different types of proteinases examined, MMP‐1, ‐3, and ‐7, cathepsin G and leukocyte elastase could digest tenascin, but MMP‐2, MMP‐9 and thrombin did not. This suggests that tenascin may be readily catabolized by extracellular matrix‐degrading proteinases found in the pathophysiological conditions.
The DNA recognition subunit of a DNA methyltransferase is predominantly a molten globule in the absence of DNA
Authors: Hornby David P. , Whitmarsh Alan , Pinarbasi Hatice , Kelly Sharon M. , Price Nicholas C. , Shore Paul , Baldwin Geoffrey S. , Waltho Jonathan
Published: July 2002

Enzyme‐catalysed DNA methylation provides an opportunity for the modulation of protein‐DNA recognition in biological systems. Recently we have demonstrated that the smaller of the two subunits of the heterodimeric, cytosine‐specific DNA methyltransferase, M.AquI, is largely responsible for sequence‐specific DNA recognition. Here we present evidence from a series of NMR, fluorescence and circular dichroism spectroscopy experiments that the DNA binding subunit of M.AquI has the characteristics of a molten globule in the absence of the catalytic machinery. In this metastable state this subunit retains its ability to bind DNA in a sequence‐specific manner. We believe this finding offers an insight into the structural flexibility which underpins the mechansim of action of these enzymes, and may provide a possible biological role for molten globules in protein function.
Crystallization and preliminary X‐ray analysis of UMP/CMP‐kinase from Dictyostelium discoideum with the specific bisubstrate inhibitor P1‐(adenosine 5′)‐P5‐(uridine 5′)‐pentaphosphate (UP5A)
Authors: Wiesmüller Lisa , Scheffzek Klaus , Kliche Werner , Goody Roger S. , Wittinghofer Alfred , Reinstein Jochen
Published: July 2002

UMP/ICMP‐kinase (UK) from the slime mold Dictyostelium discoideum has been purified to high homogeneity and co‐crystallized with the bisubstrate inhibitor P1‐(adenosine 5′)‐P5‐(uridine 5′)‐pentaphosphate (UP5A). UP5A binds to UK with a dissociation constant (K

d
) of 3 ± 0.5 nM at 25°C and pH 7.5. This is some 50‐fold tighter than the binding of P1,P5‐(diadenosine 5′)‐pentaphosphate (AP5A, K

d
= 160 ± 15 nM). AP5A is a bisubstrate inhibitor that is specific for adenylate kinase. The crystals have the symmetry of the tetragonal space group P41212 or its enantiomorph P43212. The unit cell dimensions are . The crystals diffract to a Bragg spacing of 2.1 Å.