bluuebunny/crossref_metadata_2025 huggingface dataset:
Antithrombin Glasgow, 393 arg to his: A P1 reactive site variant with increased heparin affinity but no thrombin inhibitory activity
Authors: Owen Maurice C. , Beresford Charles H. , Carrell Robin W.
Published: July 2002

Antithrombin Glasgow is a hereditary abnormal antithrombin that has lost thrombin inhibitory activity. It was isolated from the plasma of a 41‐year‐old male with a history of thrombotic events. Antithrombin Glasgow was purified from plasma using heparin‐Sepharose chromatography at pH 7.4 eluting with increasing concentrations of NaCl. The normal protein eluted with 0.9 mol/l NaCl and Glasgow with 1.05 mol/l NaCl. Electrophoresis in agarose at pH 8.6 showed the variant to migrate more anodally than normal. The C‐terminal small fragment resulting from catalytic cleavage with elastase between P3 and P4 of the reactive loop was isolated and sequenced. This showed the replacement of the arginine at residue 3 by a histidine. This is residue 393 in the intact molecule. The findings suggest that heparin, on binding, interacts indirectly with the reactive centre region of antithrombin.
Sequence‐specific chemical modification of a hybrid bacteriophage M 13 single‐stranded DNA by alkylating oligonucleotide derivatives
Authors: Vlassov V.V. , Zarytova V.F. , Kutyavin I.V. , Mamaev S.V.
Published: July 2002

Alkylating oligonucleotide derivatives react with the complementary sequences in hybrid M 13mp7 bacteriophage single‐stranded DNA and destroy the infecting ability of the DNA. The reagents do not damage M 13mp9 single‐stranded DNA lacking the target nucleotide sequence.
Bovine type XII collagen: Amino acid sequence of a 10 kDa pepsin fragment from periodontal ligament reveals a high degree of homology with the chicken α1(XII) sequence
Authors: Dublet B. , Dixon E. , de Miguel E. , van der Rest M.
Published: July 2002

A 10 kDa collagenous peptide, derived from a 30 kDa disulfide bonded fragment, was purified from bovine periodontal ligament. Amino acid sequence analysis of tryptic peptides demonstrated a 92.8% homology with the chicken α1(XII) cDNA derived sequence, demonstrating for the first time the presence of type XII collagen in a mammalian species and in an adult tissue.
Stronger affinity of reticulocyte release factor than natural suppressor tRNASer for the opal termination codon
Authors: Mizutani Takaharu , Hitaka Teruaki
Published: July 2002

Animal natural suppressor tRNA did not affect the release reaction of reticulocyte release factor (RF) at the same concentration of tRNA (both estimated as being present at a similar level of 3–5 × 10−8 M in vivo); even at a 10‐fold greater concentration the tRNA did not prevent the release reaction with RF. In order to confirm this result, the K
a values were determined. The K
a value between RF and UGA was 1.26 × 106 M−1 and that between the suppressor tRNA and UGA amounted to 8 × 103 M−1. This result showed that RF had a 150‐fold stronger affinity than suppressor tRNA for the opal termination codon. Incorporation of phosphoserine into phosphoprotein via phosphoseryl‐tRNA was inhibited by addition of RF to the reaction mixture. These results suggest that animal natural suppressor tRNA in the normal state does not perform its suppressor function, except in special cases where mRNA has the context structure near the opal termination codon (UGA).
Induction of expression of protein disulphide‐isomerase during lymphocyte maturation stimulated by bacterial lipopolysaccharide
Authors: Paver Jan L. , Freedman Robert B. , Michael R. , Parkhouse E.
Published: July 2002

Protein disulphide‐isomerase (PDI) activity, and the level of immunodetectable PDI protein, were monitored in splenic lymphocytes and in BCL1 cells during culture in the presence of various activating factors. Bacterial lipopolysaccharide stimulated induction of PDI in splenic B cells and BCL1 cells. The time‐course and specificity of induction indicated that the increase in expression of PDI is closely coupled to the final stages of B cell differentiation into antibody‐producing plasma cells. The system will prove valuable in studies on the control of expression of PDI.
Identification of both Gi2 and a novel, immunologically distinct, form of Go in rat myometrial membranes
Authors: Milligan Graeme , Tanfin Zahra , Goureau Olivier , Unson Cecilia , Harbon Simone
Published: July 2002

Immunoblotting of rat myometrial membranes with an antiserum (SG1) which recognizes the α‐subunits of both Gi1 and Gi2 indicated the presence of detectable levels of an apparently single form of some 40 kDa. A second antiserum (LE2) specific for Gi2 also recognized this protein, confirming its identity. Immunoblotting of the myometrial membranes with a series of antipeptide (OC1, IM1, ON1) and polyclonal (RV3) antisera, all of which recognize rat brain Go, produced a more complex pattern. Antisera OC1 and ON1 recognized a single polypeptide which essentially comigrated with rat brain Go. In contrast, antisera RV3 and IM1 did not recognize the myometrial protein. These data provide evidence for the presence of Gi2 and of a novel G‐protein, related immunologically to Go, in rat myometrial membranes.
A model for the dimeric molecular structure of phytochrome based on small‐angle X‐ray scattering
Authors: Tokutomi Satoru , Nakasako Masayoshi , Sakai Jun , Kataoka Mikio , Yamamoto Kotaro T. , Wada Masamitsu , Tokunaga Fumio , Furuya Masaki
Published: July 2002

The dimeric molecular structure of pea phytochrome (monomer molecular mass 114 kDa) in the red‐light‐absorbing form was studied with reference to the structures of its molecular domains in a monomer (an N‐terminal 59‐kDa chromophoric domain and a C‐terminal 55‐kDa nonchromophoric domain) using small‐angle X‐ray scattering, and a ‘four‐leaved shape’ model is proposed. The propriety of the model was confirmed by images of the molecule obtained by rotary shadowing electron microscopy.
The detection of natural opal suppressor seryl‐tRNA in Escherichia coli by the dot blot hybridization and its phosphorylation by a tRNA kinase
Authors: Mizutani Takaharu , Maruyama Naosuke , Hitaka Teruaki , Sukenaga Yoshikazu
Published: July 2002

It was believed that there was no natural suppressor tRNA in Escherichia coli, however, it has been suggested that selC, relating to the synthesis of formate dehydrogenase of a selenoprotein [(1988) Nature 331, 723–725], codes for tRNA, even though the presence of tRNA has not yet been demonstrated. We detected the product of selC in the tRNA preparation of the E. coli MC 4100 strain by the dot blot hybridization method with a DNA probe (ACCGCTGGCGGC) corresponding to the extra arm of selC tRNA. Two hybridization peaks were found in the chromatographic pattern from Sephadex A50. The amount of tRNA was estimated to be about 0.03% of the total tRNA. The suppressor [3H]seryl‐tRNA was phosphorylated by a tRNA kinase in E. coli B. These results suggest that the opal suppressor seryl‐tRNA in E. coli should be converted to selenocysteyl‐tRNA through phosphoseryl‐tRNA, and occurs in vertebrates as a general phenomenon.
A chemically transformed rat fibroblast cell line expresses high levels of oncomodulin
Authors: Sommer E.W. , Blum J.K. , Berger M.C. , Berchtold M.W.
Published: July 2002

Chemically (by N‐methyl‐N‐nitro‐N‐nitrosoguanidine) treated rat fibroblasts (T14c) exhibited growth characteristics and a morphology typical for transformed cells and markedly different from untreated, parental cells. In contrast to untransformed cells, T14c fibroblasts produced significant levels of oncomodulin mRNA as analyzed on Northern blots even when compared to rat Morris hepatomas, the richest source of oncomodulin known so far. The levels of transcripts for both calmodulin and oncomodulin in T14c cells were higher in log phase growth as compared to confluent stages.The T14c model system may be useful in the elucidation of mechanisms involved in the regulation of oncomodulin synthesis.
Differential allosteric effects of 8‐(N,N‐diethylamino)octyl‐3,4,5‐trimethoxybenzoate‐HCl (TMB‐8) on muscarinic receptor subtypes
Authors: Gordon Richard K. , Chiang Peter K.
Published: July 2002

TMB‐8, a putative inhibitor of intracellular calcium mobilization, prevents the binding of the muscarinic ligand N‐[3H]methylscopolamine. The inhibition was observed in four tissues from guinea pig; cortex, heart, pancreas, and ileum, representing Ml, cardiac M2, glandular M2, and heterogeneous M2 subtypes of muscarinic receptors, respectively. The K
i values for all four tissues were approx. 4 μM. However, dissociation kinetics revealed that TMB‐8 interacted with an allosteric site of three muscarinic receptor subtypes but not the subtype from pancreas. These results indicate that TMB‐8 interacts with muscarinic receptors, and therefore would disrupt calcium mobilization or any second messenger system coupled to these receptors.
Respiration‐dependent primary Na+ pump in halophilic marine bacterium, Alcaligenes strain 201
Authors: Kogure Kazuhiro , Tokuda Hajime
Published: July 2002

Inverted vesicle; Na+ pump; Marine bacteria; Respiratory chain
Cloning and sequence analysis of cDNA for precursor of a crustacean hyperglycemic hormone
Authors: Weidemann Wolfgang , Gromoll Jörg , Keller Rainer
Published: July 2002

Hyperglycemic hormone; Prohormone; DNA, complementary; Neuropeptide; (Carcinus maenas, Crustacean)
Recognition of functional regions in primary structures using a set of property patterns
Authors: Bork Peer
Published: July 2002

Pattern search; Property pattern; Primary structure; Recognition
The cyanelle genome of Cyanophora paradoxa encodes ribosomal proteins not encoded by the chloroplast genomes of higher plants
Authors: Bryant Donald A. , Stirewalt Veronica L.
Published: July 2002

The rpl35, rpl20, rpl5, rps8, and a portion of the rpl6 genes of the cyanelle genome of Cyanophora paradoxa have been cloned, mapped and sequenced. Homologs of the rpl35, rpl5, and rpl6 genes are not found in the chloroplasts of higher plants. The rpl35 and rpl20 genes most likely form a dicistronic operon which is located upstream from the apcE‐apcA‐apcB locus of the cyanelle and which is divergently transcribed from this locus. The rpl5, rpl8, and rpl6 genes probably form a part of a larger cluster of genes encoding components of the cyanellar ribosomes. These genes are organized in a fashion similar to that observed in all procaryotes examined to date, with the exception that the rpsl4 gene is not found between the rpl5 and rps8 coding sequences. Hypotheses concerning the origins of cyanelles and chloroplasts are discussed.
Characterization of N‐cadherin mRNA in chicken brain and heart by means of oligonucleotide probes
Authors: Dalseg Anne-Marie , Andersson Anna-Maria , Bock Elisabeth
Published: July 2002

It has previously been reported, that there is only one mRNA of 4.3 kb for chicken N‐cadherin in brain and heart and one gene [(1988) J. Cell Biol. 106, 873‐881]. Using three synthetic oligonucleotide probes derived from the published chicken N‐cadherin cDNA sequence we found hybridization to at least four mRNAs in chicken brain and heart. The measured sizes were 8.0, 4.7, 3.8, and 3.3 kb. The 8.0 kb mRNA is only seen in brain, and only when a cytoplasmic probe is employed. The 3.8 kb mRNA is only observed in heart.
Porphyrin‐linked oligonucleotides
Authors: Frolova E.I. , Ivanova E.M. , Zarytova V.F. , Abramova T.V. , Vlassov V.V.
Published: July 2002

Oligonucleotide derivatives bearing hemin and deuterohemin groups were synthesized. The derivatives efficiently react with the complementary nucleotide sequence in ssDNA forming covalent adducts and piperidine‐labile sites. In the case of the deuterohemin derivative, some direct cleavage of the target DNA occurs.
Is glucose the sole source of tissue browning in diabetes mellitus?
Authors: Hunt James V. , Wolff Simon P.
Published: July 2002

Reactions between glucose and protein are held to be responsible for the protein ‘browning’ reactions which occur in diabetes mellitus. In vitro, however, the formation of such novel protein fluorophores is dependent upon the metal‐catalysed oxidation of the monosaccharide (glucose ‘autoxidation’). Since other small oxidisable molecules are capable of ‘browning’ proteins via similar metal‐catalysed oxidative mechanisms we suggest that protein modification in diabetes may not be restricted to reactions with monosaccharides but may also include other small autoxidisable molecules.
Demonstration of a transcription element in vitro between the capping site and translation initiation site of the mouse myelin basic protein gene
Authors: Tamura Taka-aki , Mikoshiba Katsuhiko
Published: July 2002

A transcription element was identified, by in vitro analyses, just downstream from the capping site of the mouse myelin basic protein (MBP) gene. Deletion of this element caused a dramatic drop of transcription efficiency in mouse brain, rat liver and HeLa cell nuclear extracts, regardless of the form of DNA being closed circular or linear form. DNase I footprint analysis demonstrated the presence of a ubiquitous trans‐acting factor for this region. This element functioned even when it is located in the normal direction downstream from the adenovirus major late promoter. Mutation analysis suggested that an essential part of the downstream element was located between +25 and +45.
Remarkably enhanced expression of the type II hexokinase in rat hepatoma cell line AH130
Authors: Shinohara Yasuo , Ichihara Junji , Terada Hiroshi
Published: July 2002

Expression of mRNAs encoding hexokinase isozymes was studied in various cells such as rat brain, liver, skeletal muscle, kidney and heart, and the rat hepatoma cell line AH130 by Northern blotting. High specific expression of type II hexokinase was observed only with AH 130 cells. In contrast, specific expression of type I hexokinase was detected in energy‐requiring normal tissue cells such as brain and heart. These results suggest that the expression of hexokinase isozyme in the tumor cells is different from that in normal cells.
Mitochondrial preproteins en route from the outer membrane to the inner membrane are exposed to the intermembrane space
Authors: Rassow Joachim , Pfanner Nikolaus
Published: July 2002

Mitochondrial precursor proteins are known to be imported at sites of close contact between mitochondrial outer and inner membranes. We have identified translocation intermediates exposed to the intermembrane space, including the precursor of the ADP/ATP carrier accumulated at the general insertion site GIP, and the precursor of F1‐ATPase subunitβ accumulated on its import pathway at low levels of ATP. These results suggest that mitochondrial contact sites are not sealed structures, but that polypeptides pass (at least partly) through the intermembrane space on their route from the outer membrane to the inner membrane.
Analysis of a Nicotiana plumbaginifolia cDNA encoding a novel small GTP‐binding protein
Authors: Terryn N. , Anuntalabhochai S. , Van Montagu M. , Inzé D.
Published: July 2002

Small GTP‐binding proteins belonging to the Ras superfamily have been found in evolutionarily divergent organisms. Here, we report the isolation and analysis of a cDNA encoding a putative small GTP‐binding protein, designated Rhn1, from the plant, Nicotiana plumbaginifolla. The 21.8‐kDa protein has 60% amino acid similarity with the mammalian Rab5 proteins. The Rhn1 protein is encoded by a small multigene family. Northern analysis shows the highest steady‐state mRNA levels to be in roots and flowers. Furthermore, the Rhnl protein has 80% amino acid similarity with an Arabidopsis small GTP‐binding protein, designated Rha1.
Detection of a near infra‐red absorption band of ferrohaem a3 in cytochrome c oxidase
Authors: Rich Peter R. , Moody A.John , Ingledew W.John
Published: July 2002

We have detected weak absorption bands in the near infra‐red region of reduced mammalian cytochrome c oxidase, analogous to those that we have recently reported to be present in the bacterial cytochrome o (Ingledew, W.J., Bacon, M. and Rich, P.R. (1992) FEBS Lett. 305, 167–170). The major band is centred at 784 nm and has an εmM1cm1 of around 0.1. It is shifted to 760 nm in the carbon monoxide compound and is absent in the reduced cyanide complex. We attribute it to a charge transfer band of ferrohaem a3
, equivalent to the ‘band III’ or ‘conformational band’ of haemoglobin.
Unusual effects of benzodiazepines and cyclodiene insecticides on an expressed invertebrate GABAA receptor
Authors: Zaman Shahid H. , Harvey Robert J. , Barnard Eric A. , Darlison Mark G.
Published: July 2002

We have previously reported [(1991) EMBO J. 10, 3239–3245] the sequence of an invertebrate γ‐aminobutyric acid (GABA) type A (GABAA) receptor polypeptide which forms homo‐oligomeric GABA‐gated, bicuculline‐sensitive, chloride‐ion channels upon heterologous expression. We now demonstrate that the benzodiazepines Ro5‐4864 (4′‐chlorodiazepam) and diazepam, that are active at mammalian peripheral benzodiazepine sites, and not those benzodiazepines specific for central sites, directly activate the homo‐oligomeric receptor and evoke larger maximal responses than those elicited by GABA. In addition, members of the cyclodiene class of insecticides block the channel of the receptor in a manner indistinguishable from that of picrotoxin.
Loss of transcriptional repression contributes to the ectopic expression of the calcitonin/α‐CGRP gene in a human lung carcinoma cell line
Authors: Symes Aviva J. , Craig Roger K. , Brickell Paul M.
Published: July 2002

The calcitonin/α‐CORP (CT/CGRP) gene is ectopically expressed in a wide variety of neoplasia. We have investigated the molecular mechanisms responsible for this ectopic expression in the human cell line BEN, which is derived from a poorly differentiated squamous cell lung carcinoma. We show that a trans‐acting factor which represses expression of the CT/CGRP gene in HeLa cells is absent or inactive in BEN cells, and have localised the repressor binding site to a 53 bp fragment 1500 bp upstream of the transcription start site.
TATA box‐independent transcription of the human tissue plasminogen activator gene initiates within a sequence conserved in related genes
Authors: Henderson Beric R , Sleigh Merilyn J.
Published: July 2002

Transcription of the human tissue‐type plasminogen activator (tPA) gene has been reported to initiate from a single site proximal to a TATA box motif (1985, J. Biol. Chem. 260, 11223–11230). In this study, we utilized primer extension analysis to evaluate the tPA mRNA start site in phorbol‐12‐myristate 13‐acetate (PMA) induced WI‐38 man lung fibroblast cells. Whilst some tPA mRNA initiated from the predicted TATA‐proximal location (+1), a 10‐fold greater proportion of tPA mRNA transcripts initiated 110 bases downstream from a sequence conserved and utilized as the TATA‐independent transcription start site in the rodent tPA genes. Moreover, the transfection and expression in different cell types of a cosmid containing the entire human tPA gene resulted in utilization of the same downstream (+110) start site. We propose that this, rather than the previously published position, is the major transcriptional initiation point for the human tPA gene. A core sequence (5′‐CAGAGCTG‐3′) was identified which is common to the TATA‐independent mRNA start sites of the human, mouse and rat tPA genes, and which demonstrates only partial similarity to sequences found at the initiation point of other TATA‐independent genes.
Nucleocapsid protein of HIV‐1 and its Zn2+ complex formation analysis with electrospray mass spectrometry
Authors: Surovoy Andrej , Waidelich Dietmar , Jung Günther
Published: July 2002

The Zn2+ binding properties of the synthetic nucleocapsid protein (Ncp7) of HIV‐1, containing two zinc‐binding domains, have been studied using electrospray mass spectrometry (ES‐MS). ES‐MS measurements revealed strong binding of Zn2+ by Ncp7. Its shorter fragments, Ncp7‐(1–35)‐ and (29–55)‐peptides, each containing only one zinc‐binding domain, bind one equivalent of Zn2+ ions tightly. ES‐MS studies allows these fragments to be distinguished in terms of their binding affinity: they showed stronger binding of Zn2+ by Ncp7‐(1–35)‐peptide. Surprisingly, in addition to the expected two zinc‐binding domains, a third metal binding site was detected in Ncp7. However, this site appears to bind different metal ions without selectivity and most probably reflects salt formation at the C‐terminal acidic residues.
Maturation of human lactase‐phlorizin hydrolase Proteolytic cleavage of precursor occurs after passage through the Golgi complex
Authors: Lottaz D. , Oberholzer T. , Bähler P. , Semenza G. , Sterchi E.E.
Published: July 2002

Maturation of human intestinal lactase‐phlorizin hydrolase (LPH) requires that a precursor (pro‐LPH) be proteolytically processed to the mature microvillus membrane enzyme (m‐LPH). The subcellular site of this processing is unknown. Using low‐temperature experiments and brefeldin A (BFA), intracellular transport was blocked in intestinal epithelial cells. In Caco‐2 cells incubated at 18°C pro‐LPH was complex‐glycosylated but not cleaved, while at 20°C small amounts of proteolytically processed LPH were observed. These data exclude a pre‐Golgi proteolytic event. BFA completely blocked proteolytic maturation of LPH and lead to an aberrant form of pro‐LPH in both Caco‐2 cells and intestinal explants. Therefore, proteolytic processing of LPH is a post‐Golgi event, occuring either in the trans‐Golgi network, transport vesicles, or after insertion of pro‐LPH into the microvillus membrane.
Structure of UvrABC excinuclease—UV‐damaged DNA complexes studied by flow linear dichroism DNA curved by UvrB and UvrC
Authors: Takahashi Masayuki , Bertrand-Burggraf Elisabeth , Fuchs Robert P.P. , Nordén Bengt
Published: July 2002

The interaction between UvrABC excinuclease from Escherichia coli and ultraviolet light‐(UV) damaged DNA was studied by flow linear dichroism. The dichroism signal from DNA was drastically decreased in intensity upon incubation with UvrA and UvrB or whole enzyme in the presence off effector ATP. The change was specific for UV‐damaged DNA, and a concluded suppressed DNA orientation suggests the wrapping of DNA around the protein. The incubation with the UvrC subunit alone also somewhat reduces the signal, however, in this case the change was smaller and not specific for UV‐damaged DNA. The structural modification of DNA, promoted by the (UvrA2‐UvrB) complex probably facilitates or stabilizes the interaction of the UvrC subunit with DNA for the excision.
Photoreaction of N560 intermediate in the photocycle of bacteriorhodopsin
Authors: Yamamoto Noritaka , Naramoto Satoru , Ohtani Hiroyuki
Published: July 2002

Sophisticated measurements were made on the nanosecond time‐resolved absorbance change of the purple membrane of Halobacterium halobium under cw background light irradiation (440–800 nm, 11–441 mW/cm2). A red‐shifted transient species R660 (KN, Q) was found in alkaline conditions (pH > 9.3). Background light intensity effect shows that (i) R660 is photochemically formed from N560 intermediate which is accumulated under background light irradiation because of the elongated lifetime in alkaline suspension, and that (ii) the slow decaying M412 is not photochemically formed from N560 but from bR.
RNase MRP/RNase P: a structure‐function relation conserved in evolution?
Authors: Karwan Robert
Published: July 2002

RNase P and RNase MRP are related ribonucleoproteins. RNase MRP processes mitochondrial precursor‐ (primer) RNAs, whereas RNase P cleaves precursor‐tRNAs to produce their mature 5'‐ends. Both RNase P and RNase MRP are associated with the Th/To ribonucleoprotein suggesting possible interrelated pathways and/or functions. All known RNase P and RNase MRP RNAs contain conserved structural elements possibly involved in catalysis/substrate binding, but these elements do not predict all cellular functions of the RNPs.
Phosphorylation of basic fibroblast growth factor (FGF‐2) in the nuclei of SK‐Hep‐1 cells
Authors: Vilgrain Isabelle , Gonzalez Ana Maria , Baird Andrew
Published: July 2002

The subcellular fractions containing protein kinases capable of phosphorylating basic fibroblast growth factor (FGF‐2) are unknown, but having previously characterized one that is associated with the plasma membrane [1991, Mol. Endocrinol. 5, 1003‐1012] we evaluated the catalytic properties of another in the nucleus. The reaction is time (linear up to 15 min), enzyme (2,000–25,000 nuclei/ml), and substrate (K
m, 0.18 μM) dependent, and the targets serine. DNase pretreatment of nuclei decreases the incorporation of phosphate into FGF‐2 by 50% and the reaction. It is also inhibited by heparin (EC50 1 μg/ml) and spermidine (EC503μM). Calcium and cAMP have no effect. We conclude that the kinase is distinct from PKA, and PKC, and suggest that changes in glycosaminoglycan and polyamine concentrations during the cell cycle may modulate FGF‐2 phosphorylation in the nucleus, or as it is translocated to the nucleus.
Amino acid sequence surrounding the retinal‐binding site in retinochrome of the squid, Todarodes pacificus
Authors: Hara-Nishimura Ikuko , Kondo Maki , Nishimura Mikio , Hara Reiko , Hara Tomiyuki
Published: July 2002

Squid (Todarodes pacificus) retinochrome was reduced to N‐retinyl protein with borane dimethylamine and cleaved by CNBr. The retinyl peptide was then isolated by chromatography while being monitored for absorbances at 215 and 330 nm, and the N‐terminal amino acid sequence was determined to be Ser‐Lys‐Thr‐Gly‐X‐Ala‐Leu‐Phe‐Pro. This sequence was the same that we had observed at the 7th transmembrane domain of retinochrome whose structure was reported previously. During Edman degradation of the retinyl peptide, the yield of the PTH‐lysine at the second cycle was lower than those of the other PTH‐amino acids, proving that the lysine residue forms a Schiff's base with retinal (Lys‐275 in retinochrome). The amino acid sequence surrounding the retinal‐binding lysine in retinochrome greatly differed from those in a variety of known visual pigments. This fact would be associated with the difference in the photoisomerization of chromophore between retinochrome and rhodopsin. The protein structure of retinochrome is also compared with that of rhodopsin in Todarodes.
Expression of low‐affinity NGF receptor and trkB mRNA in human SH‐SY5Y neuroblastoma cells
Authors: Ehrhard Patricia B. , Ganter Ursula , Schmutz Béatrice , Bauer Joachim , Otten Uwe
Published: July 2002

We have used the human neuroblastoma cell line SH‐SY5Y as a model system to investigate the expression and regulation of the receptors for brain‐derived neurotrophic factor (BDNF), a member of the nerve growth factor (NGF) family of neurotrophins. We demonstrate that SH‐SY5Y cells express transcripts encoding the low‐affinity NGF receptor (LNGFR) and trkB, the signal transducing receptor unit for BDNF. Interaction of BDNF with SH‐SY5Y cells increased the transcription of the c‐fos gene, showing that these molecules encode functional BDNF receptors. Our findings that differentiating agents such as retinoids and cAMP analogs increased the expression of LNGFR, but decreased trkB mRNA levels, suggest that LNGFR and trkB have different roles during neuronal differentiation.
Biochemical evidence that phytochrome of the moss Ceratodon purpureus is a light‐regulated protein kinase
Authors: Algarra Patricia , Linder Stefan , Thümmler Fritz
Published: July 2002

The phytochrome gene of the moss Ceratodon purpureus (phyCer) codes for a novel phytochrome polypeptide with a predicted molecular mass of 145 kDa that has a COOH‐terminal domain which is homologous to the catalytic domain of eukaryotic protein kinases. In this paper we report the first biochemical evidence that in fact, as predicted from the gene sequence, PhyCer represents an active, light‐regulated protein kinase. In vitro phosphorylation experiments with protonemata extracts revealed the existence of a 140 kDa protein, phosphorylated in a red/far‐red light dependent manner. The binding of a polyclonal antibody directed to the protein kinase catalytic domain of PhyCer enhanced the phosphorylation of a 140 kDa band when assayed in a renaturation‐auto‐phosphorylation experiment with nitrocellulose bound protein. These findings strongly implicate that the phyCer gene product has protein kinase activity and is capable of auto‐phosphorylation. The results of the renaturation‐phosphorylation experiments were essentially the same, no matter whether protein extracts from light grown or dark adapted moss protonemata were used. Thus, phyCer expression most likely is not light regulated.
Expression of human thromboxane synthase using a baculovirus system
Authors: Yokoyama Chieko , Miyata Atsuro , Suzuki Kenji , Nishikawa Yoshiki , Yoshimoto Tanihiro , Yamamoto Shozo , Nüsing Rolf , Ullrich Volker , Tanabe Tadashi
Published: July 2002

Human thromboxane (TX) synthase (EC 5.3.99.5) was produced by the baculovirus expression system using cDNA encoding human TX synthase [(1991) Biochem. Biophys. Res. Commun. 78, 1479‐1484]. A recombinant baculovirus TXS7 was expressed in Spodoptera frugiperda Sf9 insect cells. The expressed protein was recognized by monoclonal antibody, Kon 7 raised against human TX synthase [(1990) Blood 76, 80‐85]. The recombinant TX synthase catalyzed the conversion of prostaglandin (PG) H2 to TXA2 and 12‐hydroxy‐heptadecatrienoic acid (HHT). Both conversions of PGH2 to TXA2 and HHT by the expressed TX synthase were completely inhibited by a specific TX synthase inhibitor, OKY‐046 (5 μM).
Calcium dependent activation of the NF‐AT transcription factor by p59fyn
Authors: Baldari Cosima T. , Heguy Adriana , Telford John L.
Published: July 2002

A reporter gene under the control of a T‐cell antigen receptor response element was activated in Jurkat cells by antigen receptor triggering or by a combination of phorbol myristate acetate, which activates protein kinase C, and a calcium ionophore. Both these signals were necessary for expression of the reporter gene. When co‐transfected with a construct capable of overexpressing the tyrosine kinase p59fyn, the reporter gene was activated by PMA alone. Thus p59fyn could replace the calcium ionophore but not activation of protein kinase C. The activation by p59fyn plus PMA was blocked by EGTA and by the immunosuppressant drug cyclosporin A.
Isolation and biochemical characterization of the soluble and membrane forms of folate binding protein expressed in the ovarian carcinoma cell line IGROV1
Authors: Tomassetti Antonella , Coney Leslie R. , Canevari Silvana , Miotti Silvia , Facheris Patrizia , Zurawski Vincent R. , Colnaghi Maria I.
Published: July 2002

The human ovarian carcinoma cell line, IGROV1, produces two forms of folate binding protein (FBP), the membrane form that is anchored to the cell surface by a glycosylphosphatidylinositol tail and the soluble form that is shed into the tissue culture medium. Both forms are recognized by the monoclonal antibodies MOv18 and MOv19. Here we describe their purification and biochemical characterization. The purified soluble protein appeared as a single band with an apparent M
r of 36 kDa after SDS‐PAGE, whereas the membrane form appeared as a single band with an apparent M
r of 38 kDa. The size difference between the two forms of FBP was confirmed by gel filtration of both the native and the N‐glycanase‐treated proteins. Both purified proteins had equal capacity to bind folic acid. The immunological cross‐reactivity and the folic acid binding capability of the FBPs extracted from IGROV1 gave more evidence of the possible existence of a precursor‐product relationship between them.
A mutation blocking the formation of membrane or periplasmic endogenous and exogenous c‐type cytochromes in Escherichia coli permits the cytoplasmic formation of Hydrogenobacter thermophilus holo cytochrome c552
Authors: Sambongi Yoshihiro , Crooke Helen , Cole Jeff A. , Ferguson Stuart J.
Published: July 2002

A mutant of Escherichia coli, JCB606, shown to be pleiotropically deficient in the formation of endogenous membrane and periplasmic c‐type cytochromes, synthesised the apo form of the erogenous cytochrome c
550 from Paracoccus denitrificans, but not the holo form. In contrast, a cytoplasmically located holo form of Hydrogenobacter thermophilus cytochrome c
552 was found in E. coli JCB606. These findings support the proposition that the formation of the cytoplasmic H. thermophilus cytochrome c
552 in E. coli does not involve the physiological pathway of c‐type cytochrome biosynthesis in E. coli and that the haem insertion may be uncatalysed.
Complete reconstitution of conjugation and subsequent degradation of the tumor suppressor protein p53 by purified components of the ubiquitin proteolytic system
Authors: Shkedy Dganit , Gonen Hedva , Bercovich Beatrice , Ciechanover Aaron
Published: July 2002

The wild‐type tumor suppressor protein p53 is a short‐lived protein that plays important roles in regulation of cell cycle, differentiation, and survival. Mutations that inactivate or alter the tumor suppressor activity of the protein seem to be the most common genetic change in human cancer and are frequently associated with changes in its stability. The ubiquitin system has been implicated in the degradation of p53 both in vivo and in vitro. A mutant cell line that harbors a thermolabile ubiquitin‐activating enzyme, E1, fails to degrade p53 at the nonpermissive temperature. Studies in cell‐free extracts have shown that covalent attachment of ubiquitin to the protein requires the three conjugating enzymes: E1, a novel species of ubiquitin‐carrier protein (ubiquitin‐conjugating enzyme; UBC),E2‐F1, and an ubiquitin‐protein ligase, E3. Recognition of p53 by the ligase is facilitated by formation of a complex between the protein and the human papillomavirus (HPV) oncoprotein E6. Therefore, the ligase has been designated E6‐associated protein (E6‐AP). However, these in vitro studies have not demonstrated that the conjugates serve as essential intermediates in the proteolytic process. In fact, in many cases, conjugation of ubiquitin to the target protein does not signal its degradation. Thus, it is essential to demonstrate that p53‐ubiquitin adducts serve as essential proteolytic intermediates and are recognized and degraded by the 26S protease complex, the proteolytic arm of the ubiquitin pathway. In this study, we demonstrate that conjugates of p53 generated in the presence of purified, E1, E2, E6‐AP, E6, ubiquitin and ATP, are specifically recognized by the 26S protease complex and degraded. In contrast, unconjugated p53 remains stable. The ability to reconstitute the system from purified components will enable detailed analysis of the recognition process and the structural motifs involved in targeting the protein for degradation.
Glucosylation of chimeric proteins in the cell wall of Saccharomyces cerevisiae
Authors: van Berkel M.A.A. , Caro L.H.P. , Montijn R.C. , Klis F.M.
Published: July 2002

Extension of a reporter protein with the carboxyterminal thirty amino acids of the cell wall mannoprotein α‐agglutinin of Saccharomyces cerevisiae resulted in incorporation of the chimeric protein in the cell wall. By Western analysis it was shown that the incorporated protein contained β‐1,6‐glucan similar to endogenous cell wall proteins, whereas excreted reporter protein was not glucosylated. This suggests that β‐1,6‐glucan is involved in anchoring mannoproteins in the cell wall.
Characterisation of the porin of Rhodobacter capsulatus 37b4 in planar lipid bilayers
Authors: Bishop N.D. , Lea E.J.A.
Published: July 2002

The outer membrane of Gram‐negative bacteria contains aqueous channels, porins, which aid the diffusion of small hydrophilic molecules across it. Escherischia coli, as enteric bacteria, are able to survive a hostile environment of proteases, surfactants, and drastic changes of osmotic pressure. Rhodobacter capsulatus is not an enteric bacterium and as such has not evolved to resist the same challenges. Porins, which have molecular weight of approximately 35 kDa, form trimeric channels with a solute exclusion limit of about 600 Da. Most of them open and close in a controlled manner as a function of p.d. This function is little understood at present. The functional properties of single trimers of the major porin of Rhodobacter capsulatus 37b4 have been investigated in planar artificial bilayers. On application of a suitable p.d. the observed trimer closes in approximately three equal steps. The behaviour is completely symmetrical as regards closure in response to p.d.'s of opposite polarity and is strongly cation selective.
Identification of imidazole as l‐arginine‐competitive inhibitor of porcine brain nitric oxide synthase
Authors: Mayer Bernd , Klatt Peter , Werner Ernst R. , Schmidt Kurt
Published: July 2002

Imidazole acts as a heme‐site inhibitor of nitric oxide synthase (NOS). We used this compound to investigate whether the substrate l‐arginine binds directly to the heme or to a separate domain of brain NOS. Enzyme kinetic experiments showed that imidazole enhanced the apparent K
m for l‐arginine without affecting maximal enzyme activity, and binding studies revealed that the inhibitor displaced the radioligand N
G‐nitro‐l‐[3H]arginine in a concentration‐dependent fashion. These results demonstrate that imidazole exerts its effects on NOS in an l‐arginine‐competitive manner and that the substrate site of the enzyme may be identical with the prosthetic heme group.
An Escherichia coli cyoE gene homologue in thermophilic Bacillus PS3 encodes a thermotolerant heme O synthase
Authors: Saiki Keitarou , Mogi Tatsushi , Ishizuka Morio , Anraku Yasuhiro
Published: July 2002

The cyoE gene of the Escherichia coli bo‐type quinol oxidase operon (cyo ABCDE) has been previously shown to encode heme O synthase. To demonstrate a catalytic role of a cyoE homologue (the caaE gene) in the gene cluster for caa
3‐type cytochrome c oxidase of thermophilic Bacillus PS3, we have carried out genetic complementation analysis using the chimeric operon cyo ABCD‐caaE and heme O synthase assay using the CaaE‐overproduced E. coli membranes. We found that the caaE gene encodes a thermotolerant heme O synthase which provides an intermediate for heme A biosynthesis.
Predicted topology of the N‐terminal domain of the hydrophilic subunit of the mannose transporter of Escherichia coli
Authors: Marković-Housley Zora , Balbach Jochen , Stolz Beat , Génovésio-Taverne Jean-Claude
Published: July 2002

A folding topology for the homodimeric N‐terminal domain (IIA, 2 × 14 kDa) of the hydrophilic subunit (IIABman) of the mannose transporter of E. coli is proposed. The prediction is based on (i) tertiary structure prediction methods, and (ii) functional properties of site‐directed mutants in correlation with NMR‐derived α/β secondary structure data. The 3D structure profile suggested that the overall fold of IIA is similar to that of the unrelated protein, flavodoxin, which is an open‐stranded parallel β‐sheet with a strand order of 5 4 3 1 2. The 3D model of IIA, constructed using the known atomic structure of flavodoxin, is consistent with the results from site‐directed mutagenesis. Recently NMR results confirmed the open parallel β‐sheet with a strand order of 4 3 12 (residues 1‐120) of our model whereas β‐strand 5 (residues 127–130) was shown to be antiparallel to β‐strand 4. The correctly predicted fold includes 90% of the monomeric subunit sequence and contains all functional sites of the IIA domain.
Comparison of different ribozymes for efficient and specific cleavage of BCR/ABL related mRNAs
Authors: Lange Winand , Daskalakis Michael , Finke Jürgen , Dölken Gottfried
Published: July 2002

In chronic myelogenous leukemia (CML) the reciprocal translocation of the long arms of chromosomes results in the formation of the unique BCR/ABL fusion gene which is believed to play a crucial role in the pathogenesis of CML. Different short synthetic ribozyme constructs were compared with regard to their efficiency to cleave the BCR/ABL target RNA. In the CML cell line K562 we were able to inhibit the p210BCR/ABL synthesis by a ribozyme which was about twofold more effective than the corresponding antisense molecule.
Cloning and sequencing a novel metallothionein I isoform expressed in human reticulocytes
Authors: Lambert Elizabeth , Kille Peter , Swaminathan R.
Published: July 2002

Total RNA from human reticulocytes was purified and reverse‐transcribed into cDNA using an oligo‐dT primer. This cDNA was used as a template for a polymerase chain reaction (PCR) with a primer specific for the N‐terminal sequence of mammalian metallothioneins (MT) and a universal primer. A single amplified fragment was thus generated which when cloned and sequenced revealed two distinct MT cDNAs of almost identical molecular weights. One sequence was identical to that previously reported for human MT II and the other encoded a novel MT I isoform (MT IR). The DNA sequence of MT 1R is distinct from those documented for other MT I isoforms.
Suppression of fibroblast cell growth by overexpression of LIM‐kinase 1
Authors: Higuchi Osamu , Baeg Gyeong-Hun , Akiyama Tetsu , Mizuno Kensaku
Published: July 2002

LIM‐kinase 1 (LIMK1) is a serine/threonine kinase containing two LIM motifs at the N‐terminus. The functional role of LIMKI has remained unknown. In this study, we examined the role of LIMK1 in cell growth of fibroblasts. Induced expression of LIMK1 in NIH3T3 cells led to growth retardation. Transfection of LIMK1 sense cDNA into NIH3T3 and H‐ras‐transformed FYJ10 fibroblasts significantly suppressed colony formation of these cells. In contrast, transfection with LIMK1 antisense cDNA strongly stimulated colony formation of the NIH3T3 cells. These findings suggest that LIMK1 functions as a negative regulator of fibroblast cell growth, and may play a role in tumor suppression.
Controlled ovarian hyperstimulation with or without intrauterine insemination for the treatment of unexplained infertility
Authors: Gregoriou O. , Vitoratos N. , Papadias C. , Konidaris S. , Gargaropoulos A. , Louridas C.
Published: July 2002

Objective: To evaluate whether there are any benefits from intrauterine insemination (IUI) as opposed to timed intercourse (TI) in stimulated ovarian cycles in couples with longstanding, unexplained infertility. Methods: Forty‐six couples with diagnoses of unexplained infertility were evaluated in a crossover study after a total of 141 cycles. Sixty‐seven cycles were with IUI after controlled ovarian hyperstimulation (COH) while 74 cycles were after COH and TI. Results: The pregnancy rate after COH/TI was 16.7% and after COH/IUI 45.2%. Cycle fecundity however was 8.9% after COH/TI and 25.7% after COH/IUI, which is a statistically significant difference (P < 0.05). Conclusions: A trial of human menopausal gonadotropin and IUI is justified in couples with prolonged infertility of unknown cause.
Evidence for the dual coupling of the rat neurotensin receptor with pertussis toxin‐sensitive and insensitive G‐proteins
Authors: Gailly Philippe , Najimi Mustapha , Hermans Emmanuel
Published: July 2002

We previously demonstrated the functional coupling of the rat neurotensin receptor NTS1 with G‐proteins on transfected CHO cell homogenates by showing modulation of agonist affinity by guanylyl nucleotides and agonist‐mediated stimulation of [35S]GTPγS binding. In the present study, we observed that Gi/o‐type G‐protein inactivation by pertussis toxin (PTx) resulted in a dramatic reduction of the NT‐induced [35S]GTPγS binding whereas the effect of guanylyl nucleotide was almost not affected. As expected, NT‐mediated phosphoinositide hydrolysis and intracellular calcium mobilization were not altered after PTx treatment. This suggests the existence of multiple signaling cascades activated by NT. Accordingly, using PTx and the PLC inhibitor U‐73122, we showed that both signaling pathways contribute to the NT‐mediated production of arachidonic acid. These results support evidence for a dual coupling of the NTS1 with PTx‐sensitive and insensitive G‐proteins.
Structural characterization of the circulating soluble FGF receptors reveals multiple isoforms generated by secretion and ectodomain shedding
Authors: Hanneken Anne
Published: July 2002

Soluble fibroblast growth factor receptors (FGFRs) have been identified in multiple biological fluids, including blood. Efforts to examine the biological properties of these proteins have been hampered by the incomplete chemical characterization of the receptors within the second half of the third immunoglobulin (Ig)‐like domain, where alternative splicing leads to receptor variants with different ligand binding properties. Using mass spectrometry techniques, we have mapped the soluble FGFRs to the secreted receptor, FGFR1(IIIa), the two and three Ig‐like domain isoforms of FGFR1(IIIc) and a carboxyl‐terminal cleavage peptide from the two and three Ig‐like domain isoforms of FGFR1(IIIb). The secreted FGFR is produced by the translation of an alternatively spliced transcript and the cleaved receptors are released by ectodomain shedding of the transmembrane receptors.
Multiple PIP2 binding sites in Kir2.1 inwardly rectifying potassium channels
Authors: Soom Malle , Schönherr Roland , Kubo Yoshihiro , Kirsch Cornelia , Klinger Reinhard , Heinemann Stefan H.
Published: July 2002

Inwardly rectifying potassium channels require binding of phosphatidylinositol‐4,5‐bisphosphate (PIP2) for channel activity. Three independent sites (aa 175–206, aa 207–246, aa 324–365) were located in the C‐terminal domain of Kir2.1 channels by assaying the binding of overlapping fragments to PIP2 containing liposomes. Mutations in the first site, which abolished channel activity, reduced PIP2 binding of this fragment but not of the complete C‐terminus. Point mutations in the third site also reduced both, channel activity and PIP2 binding of this segment. The relevance of the third PIP2 binding site provides a basis for the understanding of constitutively active Kir2 channels.
Role of N‐glycans and SDF‐1α on the coassociation of CD4 with CXCR4 at the plasma membrane of monocytic cells and blood lymphocytes
Authors: Mbemba Elisabeth , Saffar Line , Gattegno Liliane
Published: July 2002

CXCR4 is a coreceptor, along with CD4, for human immunodeficiency virus type 1 (HIV‐1). Trimolecular complexes between HIV‐1 glycoprotein (gp)120, CD4 and CXCR4 constitute a prerequisite for HIV entry. We studied whether CD4 is associated with CXCR4 on CD4+ CXCR4+ cells. Using the conformation‐dependent anti‐CXCR4 mAb 12G5, CD4 was coimmunoprecipitated with CXCR4 from the membrane of U937 cells which support HIV‐1LAI efficient infection, and from that of peripheral blood lymphocytes (PBL). CD4 association with CXCR4 increased upon PBL coculture for 5 days with autologous monocytes, decreased upon treatment of the cells or the CD4‐CXCR4 complex with either N‐glycanase or stromal cell derived factor‐1α (SDF‐1α) and was abolished by incubation of the cells with both, N‐glycanase and SDF‐1α. This indicates that glycans are partly involved in CD4 association with CXCR4 and may partly explain the inhibitory effect of SDF‐1α on HIV infection.
Use of phage display for isolation and characterization of single‐chain variable fragments against dihydroflavonol 4‐reductase from Petunia hybrida
Authors: De Jaeger Geert , Buys Emmanuel , Eeckhout Dominique , Bruyns Anne-Marie , De Neve Myriam , De Wilde Chris , Gerats Tom , Van Montagu Marc , Fischer Rainer , Depicker Ann
Published: July 2002

© 1997 Federation of European Biochemical Societies.
The Arabidopsis thaliana cDNA sequencing projects1
Authors: Delseny Michel , Cooke Richard , Raynal Monique , Grellet Françoise
Published: July 2002

© 1997 Federation of European Biochemical Societies.