bluuebunny/crossref_metadata_2025 huggingface dataset:
Extracellular secretion of STa heat‐stable enterotoxin by Escherichia coli after fusion to a heterologous leader peptide
Authors: Sanchez J. , Solorzano R.M. , Holmgren J.
Published: July 2002

The mature 19‐amino acid STa heat‐stable enterotoxin of E. coli has a preceding peptide of 53 amino acids which contains two domains called Pre (aa 1–19) and Pro (aa 20–53) sequences, proposed to be essential for extracellular toxin release by this host. The Pro sequence, however, has been proven not be indispensable for this process since Pro deletion mutants secrete STa. To find out if Pre and/or other unremoved natural STa flanking sequences are responsible for toxin secretion in those mutants we genetically fused mature STa directly to the leader peptide of the periplasmic E. coli heat‐labile enterotoxin B‐subunit (LTB). Expression of this gene fusion resulted in extracellular secretion of biologically active STa by E. coli independently of natural STa neighboring genetic sequences. Moreover, these results suggest that STa might be able to gain access to the extracellular milieu simply upon its entry into the E. coli periplasm once guided into this compartment by the LTB leader peptide. To test if extracellular secretion in this fashion might be extended to other disulfide bond‐rich small peptides, the 13 amino acid conotoxin GI and a non‐enterotoxic STa‐related decapeptide were cloned. None of the two peptides was found in culture supernatants, in spite of high structural homology to the toxin. Failure to be secreted most likely leads to degradation as peptides were also not detected in bacterial sonicates. We hypothesize that cysteine‐rich peptides must have an amino acid length and/or number of disulfide bridges closer to those in STa for them to follow this toxin secretory pathway in E. coli.
Tamoxifen and related compounds decrease membrane fluidity in liposomes
Authors: Wiseman Helen , Quinn Peter , Halliwell Barry
Published: July 2002

Tamoxifen and related compounds decrease membrane fluidity in ox‐brain phospholipid liposomes: their order of effectiveness is, 4‐hydroxytamoxifen > 17β‐oestradiol > tamoxifen >
cis‐tamoxifen >
N‐desmethyltamoxifen > cholesterol. A good positive correlation was demonstrated between the decrease in membrane fluidity by these compounds and their antioxidant ability as inhibitors of liposomal and microsomal lipid peroxidation (correlation coefficient, r = 0.99, P < 0.001, in both cases). The ability of tamoxifen to decrease membrane fluidity is suggested to be the mechanism of its antioxidant action and is discussed in relation to its anticancer and cardioprotective actions.
The structure of the complex between avidin and the dye, 2‐(4'‐hydroxyazobenzene) benzoic acid (HABA)
Authors: Livnah Oded , Bayer Edward A. , Wilchek Meir , Sussman Joel L.
Published: July 2002

The crystal structure of the complex formed between the egg‐white biotin‐binding protein, avidin, and the dye, 2‐(4'‐hydroxyazobenzene) benzoic acid (HABA), was determined to a resolution of 2.5 A. The interaction of avidin with the benzoate ring of HABA is essentially identical to that of the complex formed between HABA and streptavidin (the bacterial analogue of the egg‐white protein). This interaction emulates the definitive high‐affinity interaction of both proteins with the ureido moiety of biotin. The major difference between the avidin‐ and streptavidin‐HABA complexes lies in their interaction with the hydroxyphenyl ring of the dye molecule; in avidin, two adjacent amino acid residues (Phe72 and Ser73), which are not present in streptavidin, form additional interactions with this ring. These are suggested to account for the higher affinity of avidin for HABA. The characteristic red shift, which accompanies the interaction of both proteins with the dye, was traced to a proposed charge‐transfer complex formed between the hydroxyphenyl ring of HABA and the indole ring of Trp70 in avidin (Trp79 in streptavidin). Comparison of binding site residues of two such similar proteins versus their markedly different affinities for two such different substrates should eventually contribute to a better design of biomimetic reagents and drugs.
Specificity and promiscuity in membrane helix interactions
Authors: Lemmon Mark A. , Engelman Donald M.
Published: July 2002

Transmembrane α‐helices can associate with one another in lipid bilayers. This association is important in the folding and oligomerization of many integral membrane proteins, and may also play a role in their function. The interactions between helices may be highly specific or relatively non‐specific, and their roles may differ accordingly. These two cases are discussed.
One of the retinoic acid‐inducible cDNA clones in mouse embryonal carcinoma F9 cells encodes a novel isoenzyme of fructose 1,6‐bisphosphatase
Authors: Nomura Midori , Takihara Yoshihiro , Yasunaga Teruo , Shimada Kazunori
Published: July 2002

Rae‐30, one of the retinoic acid (RA)‐inducible cDNA clones in mouse embryonal carcinoma F9 cells, was sequenced and the deduced RAE‐30 protein showed about a 70% homology to mammalian fructose 1,6‐bisphosphatase (EC 3.1.3.11) (FBPase), in comparison to over 85% homology observed among the previously documented rat liver, pig kidney and human leukemic HL‐60 cell FBPases. The Rae‐30 mRNAs were not detected in various tissues of adult mice, including the liver and kidney, but were detected in a placenta and predominantly in the intestine of adult mice. These findings indicate that the Rae‐30 cDNA encodes a novel isoenzyme of FBPase, which is likely to be involved in early differentiation in mammalian cells.
A novel dehydroascorbate reductase from spinach chloroplasts homologous to plant trypsin inhibitor
Authors: Trümper Susanne , Follmann Hartmut , Häberlein Ingo
Published: July 2002

Dehydroascorbate reductase has been isolated from spinach chloroplasts and purified to apparent homogeneity. The N‐terminal amino acid sequence of the enzyme is homologous to the Kunitz‐type trypsin inhibitors from plant sources. It is shown that spinach DHA reductase and soybean trypsin inhibitor are both capable of reducing dehydroascorbate when in the reduced (thiol) form but acquire trypsin‐inhibiting activity in the oxidized (disulfide) state. Reduced chloroplast thioredoxins also reduce dehydroascorbate.
Expression of two variants of the human μ opioid receptor mRNA in SK‐N‐SH cells and human brain
Authors: Bare Lance A. , Mansson Erik , Yang Dingmming
Published: July 2002

A partial μ opioid receptor gene was isolated from a human genomic library using a mouse δ opioid receptor cDNA as a probe. Using information from this genomic clone and the published human μ receptor, MOR1 a cDNA was isolated from SK‐N‐SH mRNA that codes for a variant of the MOR1 mRNA, MOR1A. The presence of MOR1A is also shown in human brain using RT‐PCR. MOR1A differs from MOR1 in that the 3′ terminal intron has not been removed. An in‐frame termination codon is found four amino acids after the 5′ consensus splice site, making MOR1A eight amino acids shorter than MOR1. Both receptors show similar ligand binding and coupling to cAMP in CHO‐K1 cells. The C‐terminal differences between MOR1 and MOR1A could have effects on receptor coupling or receptor transport and localization.
Cloning of the rat and human prostaglandin F2α receptors and the expression of the rat prostaglandin F2α receptor
Authors: Lake S. , Gullberg H. , Wahlqvist J. , Sjögren A.-M. , Kinhult A. , Lind P. , Hellström-Lindahl E. , Stjernschantz J.
Published: July 2002

We have cloned the FP receptor from rat corpus luteum and human uterus cDNA libraries, respectively. The coding DNA sequence in the rat cDNA is 1101 bp and is similar to the mouse cDNA coding for a receptor protein of 366 amino acids. The human sequence shows a 5 bp deficiency in the 3′ region, truncating the coding sequence to 359 amino acids. Northern blot analysis indicates highest expression in the ovary. Cell lines have been established giving stable expression of the FP receptor. Activation of the cloned FP receptor gave an increase in intracellular calcium, indicating signaling via phospholipase C‐mediated phosphoinositide turnover. Using [3H]PGF2α, binding of PGs showed the rank order of fluprostenol > PhXA70 > PGF2α > ‐ PhXA85 > PGD2 > PGE2.
Retinoic acid induces BDNF responsiveness of sympathetic neurons by alteration of Trk neurotrophin receptor expression
Authors: Kobayashi Miwako , Kurihara Kenzo , Matsuoka Ichiro
Published: July 2002

The expression of high affinity neurotrophin receptors (TrkA, TrkB, and TrkC) determines the survival response of different populations of neurons to specific members of the neurotrophin family, including nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), and neurotrophin‐3 (NT‐3). However, the mechanism which controls the expression of neurotrophin receptors during neuronal development is largely unknown. Here we show that the treatment of the cultured sympathetic neurons from newborn rat superior cervical ganglia (SCG) with retinoic acid (RA), a derivative of vitamin A, suppressed the expression of trkA mRNA and induced the expression of trkB mRNA. Expression of the functional TrkB receptor was confirmed by the emergence of trophic dependence of these neurons on BDNF in the absence of NGF. Differential regulation of trk mRNAs by RA provides a possible model for the establishment of neurotrophin dependence of peripheral neurons.
A well resolved ODMR triplet minus singlet spectrum of P680 from PSII particles
Authors: Carbonera Donatella , Giacometti Giovanni , Agostini Giancarlo
Published: July 2002

An ADMR T‐S spectrum of the primary donor (P680) of photosystem II (PSII) was obtained from anaerobically photoreduced particles. The spectrum is the best resolved obtained so far having a main bleaching band at 684 nm with a linewidth of only 100 cm−1. The view that this spectrum is produced by native homogeneous P680 unlike those obtained before is defended. A small bleaching observed at 678 nm is discussed in terms of the reaction center structure. One possible interpretation of the observations is that P680 is a very loose dimer with an exciton splitting of only 144 cm−1 corresponding to a dimer center‐to‐center distance of roughly 11.5 Å.
Determination of the number of detergent molecules associated with the reaction center protein isolated from the photosynthetic bacterium Rhodopseudomonas viridis
Authors: Gast P. , Hemelrijk P. , Hoff A.J.
Published: July 2002

Detergent‐free reaction center (RC) proteins from the photosynthetic bacterium Rhodopseudomonas viridis were obtained using Bio‐Beads SM‐2. With these RCs, the amount of detergent molecules associated with the protein was measured by determining the detergent concentration at which re‐solubilization occurred as a function of the RC concentration. For N,N dimethyl dodecylamine‐N‐oxide (LDAO), Triton X‐100 and β‐octylglucoside 260 ± 30,105 ± 10 and 360 ± 100 detergent molecules were necessary to dissolve the protein, respectively. With this technique we have studied the effect of the amphiphilic molecule 1,2,3‐heptanetriol, which is essential in the crystallization process of these RCs. Addition of 5% 1,2,3‐heptanetriol reduces the value for LDAO to 120 ± 20 LDAO/RC, supporting the notion that crystallization of the RCs is promoted by increasing the number of protein‐protein contacts.
Intracellular free calcium level and its response to cAMP stimulation in developing Dictyostelium cells transformed with jellyfish apoaequorin cDNA
Authors: Saran Shweta , Nakao Hajime , Tasaka Masao , Iida Hidetoshi , Tsuji Frederick I. , Nanjundiah Vidyanand , Takeuchi Ikuo
Published: July 2002

A new method is described for measuring intracellular free calcium concentrations, [(Ca2+
i)], in the cells of Dictyostelium discoideum transformed with apoaequorin cDNA of the jellyfish, Aequorea victoria. Aequorin, a calcium‐specific indicator, was regenerated in vivo from apoaequorin produced in the cells by incubation with coelenterazine. The results showed that [(Ca2+)i] in developing cells markedly increases at the aggregation stage and again at the culmination stage after a temporary drop at the migration stage. Except for the vegetative stage, the cells at all stages of development exhibit a sharp transient increase in [(Ca2+)i] upon stimulation with a cAMP (50 nM) pulse, high responses being observed at the migration and culmination stages. Separated prestalk cells of migrating slugs contain more than twice as much [(Ca2+)i] and show three times as large a response to cAMP stimulation as prespore cells.
Role of heterotrimeric GTP binding proteins in vesicular protein transport: indications for both classical and alternative G protein cycles
Authors: Helms J.Bernd
Published: July 2002

Heterotrimeric G proteins are involved in hormonal signal transduction across the plasma membrane. Recent evidence suggests that they have a role in vesicular protein transport as well. Biochemical probes that interfere with the classical G protein cycle have been applied to the field of intracellular membrane transport to study their mechanism of action. Evidence has been obtained that intracellular G proteins act both through classical and alternative G protein cycles.
The rate of thermal inactivation of Torpedo acetylcholinesterase is not reduced in the C231 S mutant
Authors: Wilson Erica J. , Massoulié Jean , Bon Suzanne , Rosenberry Terrone L.
Published: July 2002

The rate of thermal inactivation of Torpedo AChE at pH 8.5 was increased by the sulfhydryl reagent 5,5′‐dithiobis‐(2‐nitrobenzoic acid) (DTNB). At 30°C or 37°C, inactivation rates with 0.3 mM DTNB increased about 5‐fold for the wild‐type enzyme and for two site‐specific mutants, D72S and V129R. The reversible active site inhibitor, ambenonium, completely stabilized the wild type enzyme and partially stabilized the D72S mutant. However, ambenonium did not protect against the destabilization introduced by DTNB, which still accelerated inactivation of D72S 5‐fold. When the only free sulfhydryl group in AChE was removed by replacing cysteine 231 with serine, increased rates of thermal inactivation were observed. The inactivation rate incrased by a factor of 2 to 3 for the single mutant (C231S) and by a factor of 5 for the double mutant V129R/C231S. Even in the C231S mutants, DTNB still had an additional effect. It increased the inactivation rate for C231S and V129R/C231 by a factor of about 1.5 to 3 beyond the rates seen in the absence of DTNB. Therefore, at least part of the destabilization seen with DTNB in enzymes that retain C231 does not involve reaction of DTNB with C231.
Regulation of type V adenylyl cyclase by PMA‐sensitive and ‐insensitive protein kinase C isoenzymes in intact cells
Authors: Kawabe Jun-ichi , Ebina Toshiaki , Toya Yoshiyuki , Oka Naoki , Schwencke Carsten , Duzic Emir , Ishikawa Yoshihiro
Published: July 2002

Type V adenylyl cyclase (AC) was stably over‐expressed in HEK293 cells (293AC‐V). Forskolin‐stimulated cAMP accumulation in 293AC‐V was 5 times as great as that in control cells. PMA, a protein kinase C (PKC) activator, enhanced cAMP accumulation in 293AC‐V cells dose‐and time‐dependently and this enhancement was abolished by staurosporine. Insulin also enhanced cAMP accumulation in 293AC‐V cells. Co‐transfection of PKC‐ζ, but not PKC‐α, potentiated the effects of insulin. These data suggest that type V AC activity is regulated in cells by PKC isoenzymes through different extracellular stimuli.
The respiration‐driven active sodium transport system in E. coli does not function with lithium
Authors: Verkhovskaya Marina L. , Verkhovsky Michael I. , Wikström Mårten
Published: July 2002

Comparison of respiration‐driven active transport of alkali cations from E. coli cells loaded with Na+ or Li+ showed that Li+ could not be expelled from the cells like Na+. K+ accumulation, which was fast in Na+‐loaded cells, was strongly inhibited in Li+‐loaded cells, despite high membrane potential and respiratory rate. When Li+‐loaded cells were placed into medium containing Na+ instead of Li+, Li+/Na+ exchange took place initially, while K+ accumulation was delayed. Only after almost all inside Li+ was replaced by Na+ did active Na+ and K+ transport commence. These data confirm that it is a distinct active sodium transport system (AST) with Na+,K+/H+ antiporter activity, and not the Na+/H+ antiporters, that is responsible for active Na+ transport in E. coli [Verkhovskaya, M.L., Verkhovsky, M.I. and Wikström, M. (1996) Biochim. Biophys. Acta 1273, 207–216]. In contrast to the Na+/H+ antiporters, the AST system is inhibited by Li+.
Membranotropic properties of the water soluble amino acid and peptide derivatives of fullerene C60
Authors: Kotelnikova Raisa A. , Kotelnikov Alexander I. , Bogdanov Guennady N. , Romanova Valentina S. , Kuleshova Elena F. , Parnes Zinaida N. , Vol'pin Mark E.
Published: July 2002

The modifying effects of the products of the equimolar addition of dl‐alanine and dl‐alanyl‐dl‐alanine to fullerene C60 on the structure and permeability of the lipid bilayer of phosphatidylcholine liposomes has been studied using the luminescence probe technique. It is shown that these water soluble amino acid and dipeptide derivatives of fullerene (C60‐AD) are quenchers of pyrene fluorescence and erythrosine phosphorescence of in both a water solution and liposomes. To study the permeability of the lipid bilayer a procedure based on the triplet probe technique has been developed. It has been found that the C60‐AD derivatives under study are able to localize inside the artificial membrane, to penetrate into the liposomes through the lipid bilayer and to perform activated transmembrane transport of bivalent metal ions.
Calmodulin inhibits calcium influx current in vascular endothelium
Authors: Vaca Luis
Published: July 2002

Utilizing the whole‐cell configuration of the patch‐clamp technique the effect of calmodulin (CaM) on thapsigargin‐induced Ca2+ current has been studied. Addition of several concentrations of CaM to the patch pipette induced concentration‐dependent inhibition of thapsigargin‐induced Ca2+ current in bovine aortic endothelial cells. The effect of CaM was Ca2+ dependent and was not observed when the intracellular Ca2+ was buffered to 1 nM with EGTA. CaM produced two major effects on the thapsigargin‐induced Ca2+ current. First CaM slow down activation of the current by thapsigargin from a control value of 16 ± 5 to 31 ± 6 s with 1 μM CaM in the pipette solution. The second effect of CaM was to reduce the current amplitude in a concentration‐dependent manner. The inhibition of Ca2+ current was observed at the peak of the current and at the sustained current level. The reduction of current at the sustained level was observed 15–20 s after onset of the thapsigargin response. The half inhibitory concentration determined from these experiments was 0.1 μM. These results indicate that CaM can modulate thapsigargin‐induced Ca2+ current in this endothelium, suggesting a possible role for CaM in the regulation of store‐operated Ca2+ influx.
Four Arabidopsis thaliana 14‐3‐3 protein isoforms can complement the lethal yeast bmh1bmh2 double disruption
Authors: van Heusden G.Paul H. , van der Zanden A.Linda , Ferl Robert J. , Steensma H.Yde
Published: July 2002

The 14‐3‐3 proteins comprise a family of highly conserved proteins with multiple functions, most of which are related to signal transduction. Four isoforms from the plant Arabidopsis thaliana were able to complement the lethal disruption of the two Saccharomyces cerevisiae genes encoding 14‐3‐3 proteins; one complemented very poorly and one did not complement. However, the expression of the latter two isoforms was very low. These results show that at least four of the six A. thaliana isoforms are able to perform the same function(s) as the yeast 14‐3‐3 proteins.
Hydatidiform mole: clinical analysis of 310 patients
Authors: Mungan T. , Kuşçu E. , Dabakoǧlu T. , Şenöz S. , Uǧur M. , Çobanoglu Ö.
Published: July 2002

Objectives: The aim of the study was to analyze the clinical characteristics, treatment and outcome of 310 patients with hydatidiform mole. Methods: Three hundred ten patients with hydatidiform mole admitted to Dr Zekai Tahir Burak Women's Hospital between 1989 and 1994, were evaluated retrospectively according to their clinical characteristics, treatment modalities and follow‐up. Results: The incidence of molar pregnancy was 2.48 per 1000 deliveries and 1.84 per 1000 pregnancies. The age of the patients ranged from 14 to 45 years with a mean age of 25.29 ± 7.40 years. In 60% of the patients, the molar pregnancy was their first pregnancy. A history of previous hydatidiform mole was found in 5.5% of the patients and eight of them had at least two previous molar pregnancies. The most common presenting symptom was vaginal bleeding (71%). Although theca‐lutein cysts were found in 17.1% of the patients, only one patient underwent emergency surgery because of torsion. Dilatation and suction curettage was the first‐line treatment; uterine perforation developed in two patients (0.6%). During follow‐up 14.5% of patients were diagnosed as persistent cases according to serum β‐human chorionic gonadotropin (β‐hCG) levels. Complete remission was achieved with the administration of 2–8 courses of single‐agent chemotherapy in 43 cases; combined chemotherapy (3–7 courses) was given to two patients who were resistant to single‐agent therapy. Conclusion: Comparison of patients with spontaneous remission and patients with persistent trophoblastic disease with respect to age, histologic type, previous history, initial uterine size, gravidity, presence of theca‐lutein cysts and initial β‐hCG levels did not reveal any of the above criteria to be prognostic for the occurrence of persistent disease.
Transvaginal sonography and hysteroscopy in women with postmenopausal bleeding
Authors: Haller H. , Matejčić N. , Rukavina B. , Krašević M. , Rupčić S. , Mozetič D.
Published: July 2002

Objective: To make a prospective comparison between endometrial thickness determined by transvaginal sonography (TVS) and hysteroscopic findings in women with postmenopausal bleeding with histologic findings obtained by dilatation and curettage (D&C). Methods: Eighty‐one patients who had not received hormonal replacement therapy were scanned by transvaginal probe, and double‐layer endometrial thickness was measured 1 day before hysteroscopy and D&C. Results: The histologic diagnosis was atrophy in 12 cases, irregular proliferative changes in 21, endometrial polyps in 16, hyperplasia in 16 and endometrial carcinoma in 16. TVS detected 46 of 48 pathologic conditions, including all cases of endometrial carcinoma if the endometrial thickness (both layers) was ≥ 5 mm (sensitivity 95.8%, specificity 45.5%). Hysteroscopy also detected the endometrial pathology in 46 of 48 cases but with a higher specificity (sensitivity 95.3%, specificity 93.9%). Conclusion: TVS and hysteroscopy are complementary diagnostic methods and could be accurately used to discriminate between normal and pathologic conditions in patients with postmenopausal bleeding.
Withdrawal from benzodiazepines by initially switching to zopiclone
Authors: Shapiro CM , Sherman D , Peck DF
Published: July 2002

Withdrawal from hypnotics can produce a variety of problems, especially sleep difficulties, some of which may arise from the multiple actions of most hypnotics, thus producing a range of rebound effects. This study examined whether switching patients to a hypnotic with a narrower range of action and of a different class would reduce these problems. One hundred and thirty-four patients participated; they were randomly allocated to one of three methods of switching from “previous hypnotic” to zopiclone (a cyclopyrrolone). The methods were gap (an interval between taking the two drugs); abuttal (taking zopiclone immediately on stopping previous drug); and overlap (gradually reducing previous drug after starting zopiclone). The main findings were that zopiclone was associated with better sleep and increased alertness; the abuttal method was the best method of switching; and no serious side effects from zopiclone were reported. It was concluded that zopiclone has a useful role in benzodiazepine withdrawal, and that immediate substitution is the best method.
Psychosocial rehabilitation of schizophrenic patients in France
Authors: Singer L , Kapfer T
Published: July 2002

The authors examine the situation of rehabilitation of schizophrenic patients in France. After describing the methodology of their survey, the results are given. The housing facilities include halfway houses, “therapeutic” flats. The rehabilitation facilities include outpatient clinics with vocational units and rehabilitation centres such as vocational centres and sheltered workshops. In addition, a few centres provide placement and follow-up in the workplace. This type of centre is a recent innovation which helps stabilized schizophrenic patients find a regular job. The Fondation Santé des Étudiants de France (the health foundation of French students) deserves particular mention because it provides rehabilitation to high school and university students. In the discussion, the authors emphasize the insufficient number of such facilities, due to French psychiatrists only quite recently becoming aware of the importance of rehabilitation, which in turn, leads to insufficient funding of public mental health. As a result, most of the rehabilitation facilities were created and are now managed by private associations such as Les Croix Marines, UNAFAM, MGEN, Fondation Santé des Étudiants, Route Nouvelle Alsace, CAPPA Clermont-Ferrand, etc. In addition, their administrative tutelage is so complex that very few new facilities are developed. In view of their experience, the authors suggest that a large number of vocational centres should be built since they provide an essential service. Thus schizophrenic patients could enjoy a decent life in our society and not become one of the already many homeless. The European Union and the Council of Europe ought to consider the increase of such centres as a significiant aspect of their mental health policy.
Purification of the 45 kDa, membrane bound NADH dehydrogenase of Escherichia coli (NDH‐2) and analysis of its interaction with ubiquinone analogues
Authors: Björklöf Katja , Zickermann Volker , Finel Moshe
Published: July 2002

The NADH:ubiquinone reductase (NDH‐2) of Escherichia coli was expressed as a His‐tagged protein, extracted from the membrane fraction using detergent and purified by chromatography. The His‐tagged NDH‐2 was highly active and catalyzed NADH oxidation by ubiquinone‐1 at rates over two orders of magnitude higher than previously reported. The purified, His‐tagged NDH‐2, like native NDH‐2, did not oxidize deamino‐NADH. Steady‐state kinetics were used to analyze the enzyme's activity in the presence of different electron acceptors. High V
max and low K
m values were only found for hydrophobic ubiquinone analogues, particularly ubiquinone‐2. These findings strongly support the notion that NDH‐2 is a membrane bound enzyme, despite the absence of predicted transmembrane segments in its primary structure. The latter observation is in agreement with possible evolutionary relation between NDH‐2 and water‐soluble enzymes such as dihydrolipoamide dehydrogenase. There is currently no clear indication of how NDH‐2 binds to biological membranes.
Identification of syntenin and other TNF‐inducible genes in human umbilical arterial endothelial cells by suppression subtractive hybridization
Authors: Stier Sebastian , Totzke Gudrun , Grünewald Elisabeth , Neuhaus Thomas , Fronhoffs Stefan , Sachinidis Agapios , Vetter Hans , Schulze-Osthoff Klaus , Ko Yon
Published: July 2002

Endothelial cells play an important regulatory role in inflammatory responses by upregulating various proinflammatory gene products including cytokines and adhesion molecules. A highly potent mediator of this process is tumor necrosis factor‐α (TNF). In the present study, the suppression subtractive hybridization (SSH) method was employed to identify rarely transcribed TNF‐inducible genes in human umbilical arterial endothelial cells. Following mRNA isolation of non‐stimulated and TNF‐stimulated cells, cDNAs of both populations were prepared and subtracted by suppression PCR. Sequencing of the enriched cDNAs identified 12 genes differentially expressed including vascular cell adhesion molecule‐1, monocyte chemoattractant protein‐1, interleukin‐8 and IκBα, an inhibitor of the transcription factor nuclear factor‐κB. Interestingly, also syntenin, a PDZ motif‐containing protein which binds to the cytoplasmic domain of syndecans, was identified by SSH. Time course studies using RT‐PCR analysis confirmed that all genes were differentially expressed and rapidly induced by TNF. Our data reveal that SSH is a powerful technique of high sensitivity for the detection of differential gene expression in primary arterial endothelial cells.
The gene encoding DRAP (BACE2), a glycosylated transmembrane protein of the aspartic protease family, maps to the Down critical region
Authors: Acquati F. , Accarino M. , Nucci C. , Fumagalli P. , Jovine L. , Ottolenghi S. , Taramelli R.
Published: July 2002

We applied cDNA selection methods to a genomic clone (YAC 761B5) from chromosome 21 located in the so‐called ‘Down critical region’ in 21q22.3. Starting from human fetal heart and brain mRNAs we obtained and sequenced several cDNA clones. One of these clones (Down region aspartic protease (DRAP), named also BACE2 according to the gene nomenclature) revealed a striking nucleotide and amino acid sequence identity with several motifs present in members of the aspartic protease family. In particular the amino acid sequences comprising the two catalytic sites found in all mammalian aspartic proteases are perfectly conserved. Interestingly, the predicted protein shows a typical membrane spanning region; this is at variance with most other known aspartic proteases, which are soluble molecules. We present preliminary evidence, on the basis of in vitro translation studies and cell transfection, that this gene encodes a glycosylated protein which localizes mainly intracellularly but to some extent also to the plasma membrane. Furthermore DRAP/BACE2 shares a high homology with a newly described β‐secretase enzyme (BACE‐1) which is a transmembrane aspartic protease. The implications of this finding for Down syndrome are discussed.
Elucidation of determinants of protein stability through genome sequence analysis
Authors: Chakravarty Suvobrata , Varadarajan Raghavan
Published: July 2002

Sequences of putative soluble proteins from complete genomes of eight thermophiles and 12 mesophiles were analyzed to gain insight into determinants of protein thermostability. The predator algorithm was used to assign secondary structures to each protein sequence. Based on simple statistical tests, a set of stabilizing factors was identified. These include reduced protein size, increases in number of residues involved in hydrogen bonding, β‐strand content and helix stabilization through ion pairs. There are also significant increases in the relative amounts of charged and hydrophobic β‐branched amino acids and decreases in uncharged polar amino acids in proteins from thermophiles relative to mesophilic organisms. Factors such as the relative proportion of residues in loops, proline and glycine content and helix capping do not appear to be important.
Tubulin folding cofactor D is a microtubule destabilizing protein
Authors: Martı́n Lara , Fanarraga Mónica L , Aloria Kerman , Zabala Juan C
Published: July 2002

A rapid switch between growth and shrinkage at microtubule ends is fundamental for many cellular processes. The main structural components of microtubules, the αβ‐tubulin heterodimers, are generated through a complex folding process where GTP hydrolysis [Fontalba et al. (1993) J. Cell Sci. 106, 627–632] and a series of molecular chaperones are required [Sternlicht et al. (1993) Proc. Natl. Acad. Sci. USA 90, 9422–9426; Campo et al. (1994) FEBS Lett. 353, 162–166; Lewis et al. (1996) J. Cell Biol. 132, 1–4; Lewis et al. (1997) Trends Cell Biol. 7, 479–484; Tian et al. (1997) J. Cell Biol. 138, 821–823]. Although the participation of the cofactor proteins along the tubulin folding route has been well established in vitro, there is also evidence that these protein cofactors might contribute to diverse microtubule processes in vivo [Schwahn et al. (1998) Nature Genet. 19, 327–332; Hirata et al. (1998) EMBO J. 17, 658–666; Fanarraga et al. (1999) Cell Motil. Cytoskel. 43, 243–254]. Microtubule dynamics, crucial during mitosis, cellular motility and intracellular transport processes, are known to be regulated by at least four known microtubule‐destabilizing proteins. OP18/Stathmin and XKCM1 are microtubule catastrophe‐inducing factors operating through different mechanisms [Waters and Salmon (1996) Curr. Biol. 6, 361–363; McNally (1999) Curr. Biol. 9, R274–R276]. Here we show that the tubulin folding cofactor D, although it does not co‐polymerize with microtubules either in vivo or in vitro, modulates microtubule dynamics by sequestering β‐tubulin from GTP‐bound αβ‐heterodimers.
Sulphur islands in the Escherichia coli genome: markers of the cell's architecture?
Authors: Rocha Eduardo P.C. , Sekowska Agnieszka , Danchin Antoine
Published: July 2002

Two highly contrasted images depict genomes: at first sight, genes appear to be distributed randomly along the chromosome. In contrast, their organisation into operons (or pathogenicity islands) suggests that, at least locally, related functions are in physical proximity. Analysis of the codon usage bias in orthologous genes in the genome of bacteria which diverged a long time ago suggested that some physical (architectural) selection pressure organised the distribution of genes along the chromosome. The metabolism of highly reactive species such as sulphur‐containing molecules must be compartmentalised to escape the deleterious actions of diffusible reagents such as gases or radicals. We analysed the distribution of sulphur metabolism genes in the genome of Escherichia coli and found a number of them to be clustered into statistically significant islands. Another interesting feature of these genes is that the proteins they encode are significantly deprived of cysteine and methionine residues, as compared to the bulk proteins. We speculate that this clustering is associated to the organisation of sulphur metabolism proteins into islands where the sensitive sulphur‐containing molecules are protected from reacting with elements in the environment such as dioxygen, nitric oxide or radicals.
AKAP79 and the evolution of the AKAP model
Authors: Dodge Kimberley , Scott John D
Published: July 2002

A molecular explanation for the specificity of the cAMP‐dependent protein kinase (PKA) can be provided by its compartmentalization through association with A‐kinase‐anchoring proteins (AKAPs). Structural and functional studies have led to the development of an anchoring model proposing that AKAPs contain a common PKA binding domain and a unique subcellular targeting domain. The discovery that AKAPs can bind other signaling enzymes led to the addition of a third property, that of scaffolding molecule. Recent research has now expanded the role of AKAPs to members of multiunit complexes containing both upstream activators and downstream targets.
A common binding site for substrates and protons in EmrE, an ion‐coupled multidrug transporter
Authors: Yerushalmi Hagit , Schuldiner Shimon
Published: July 2002

EmrE is an Escherichia coli 12‐kDa multidrug transporter, which confers resistance to a variety of toxic cations by removing them from the cell interior in exchange with two protons. EmrE has only one membrane‐embedded charged residue, Glu‐14, that is conserved in more than 50 homologous proteins and it is a simple model system to study the role of carboxylic residues in ion‐coupled transporters. We have used mutagenesis and chemical modification to show that Glu‐14 is part of the substrate binding site. Its role in proton binding and translocation was shown by a study of the effect of pH on ligand binding, uptake, efflux and exchange reactions. We conclude that Glu‐14 is an essential part of a binding site, common to substrates and protons. The occupancy of this site is mutually exclusive and provides the basis of the simplest coupling of two fluxes.
The activation of eukaryotic initiation factor (eIF)2B by growth factors in PC12 cells requires MEK/ERK signalling
Authors: Kleijn Miranda , Proud Christopher G
Published: July 2002

Epidermal and nerve growth factors (EGF and NGF) activate protein synthesis and initiation factor eIF2B in rat phaeochromocytoma (PC12) cells. The activation of protein synthesis by EGF or NGF depends upon extracellular regulated kinase kinase (MEK)/extracellular regulated kinase signalling. Here we show that PD98059, an inhibitor of MEK activation, blocks the activation of eIF2B by EGF or NGF. It is known that eIF2B activity can be inhibited by phosphorylation at Ser535 in its ϵ‐subunit by glycogen synthase kinase (GSK)‐3. We find that inactivation of GSK‐3 by EGF or NGF is blocked by PD98059. However, neither EGF nor NGF caused a detectable change in phosphorylation of Ser535 of eIF2Bϵ. Thus, the EGF‐ and NGF‐induced activation of eIF2B in PC12 cells involves regulatory mechanisms distinct from dephosphorylation of the GSK‐3 site.
Sequence and electrophysiological characterization of two insect‐selective excitatory toxins from the venom of the Chinese scorpion Buthus martensi
Authors: Escoubas Pierre , Stankiewicz Maria , Takaoka Tomoyo , Pelhate Marcel , Romi-Lebrun Régine , Wu Fang Qi , Nakajima Terumi
Published: July 2002

The two insecticidal peptides Bm32‐VI and Bm33‐I, isolated from the venom of the Chinese scorpion Buthus martensi induce paralytical symptoms typical of insect contractive toxins. They show, respectively, 74% and 77% homology with AaIT from Androctonus australis, comparable insecticidal activity and no vertebrate toxicity. Under voltage‐clamp conditions, both toxins induced (1) an increased fast Na+ current, (2) a shift in voltage dependence of Na+ current activation, (3) the occurrence of a delayed current, and (4) a slow development of a holding current. Increased Na+ conductance at negative potential values is responsible for axonal hyperexcitability and the contractive paralysis of insect prey.
A naturally occurring bacterial Tat signal peptide lacking one of the ‘invariant’ arginine residues of the consensus targeting motif
Authors: Hinsley Andrew P. , Stanley Nicola R. , Palmer Tracy , Berks Ben C.
Published: July 2002

Currently described substrates of the bacterial Tat protein transport system are directed for export by signal peptides containing a pair of invariant arginine residues. The signal peptide of the TtrB subunit of Salmonella enterica tetrathionate reductase contains a single arginine residue but is nevertheless able to mediate Tat pathway transport. This naturally occurring example of a Tat signal peptide lacking a consensus arginine pair expands the range of sequences that can target a protein to the Tat pathway. The possible implications of this finding for the assembly of electron transfer complexes containing Rieske proteins in plant organelles are discussed.
Sequence analyses and comparative modeling of fly and worm fibroblast growth factor receptors indicate that the determinants for FGF and heparin binding are retained in evolution
Authors: Nagendra H.G , Harrington Adrian E , Harmer Nicholas J , Pellegrini Luca , Blundell Tom L , Burke David F
Published: July 2002

The presence of a large number of fibroblast growth factors (FGFs) and multiple splice forms of their receptors (FGFRs) in higher vertebrates makes the three‐dimensional (3D) analysis of FGF interactions with their receptors a formidable task. The situation differs in Caenorhabditis elegans (worm) and Drosophila melanogaster (fruit fly), where only one or two FGF and FGFR sequences have been identified. Structural studies of the FGF–FGFR complexes in such primitive organisms should reveal the basic features of the ligand–receptor interactions as they first emerged through evolution. We have analysed the sequences of worm and fly FGFs and FGFRs and used the recently determined crystal structure of the human FGF1–FGFR2–heparin ternary complex [Pellegrini, L., Burke, D.F., von Delft, F., Mulloy, B. and Blundell, T.L. (2000) Nature 407, 1029–34] to construct 3D models of the homologous complexes. In spite of a low sequence similarity with their human counterparts, key structural features required for ligand–receptor and protein–heparin binding in humans are conserved in the fly and worm FGF–FGFR–heparin complexes. Analyses of the models show that tertiary interactions that are not conserved in sequence are maintained through novel interactions or complementary mutations in the fly and worm sequences. The overall charge distributions observed in the human FGF–FGFR–heparin complex are retained in the fly and worm models. The arginine residue at position 253 in the linker region between the Ig‐like domains D2 and D3 in the wild type fly and worm sequences is particularly striking, as the Pro253Arg mutation in humans is responsible for Apert syndrome. This change may enhance the affinity of receptors for their FGF molecules as observed in Apert mutants.
The SUPERMAN protein is an active repressor whose carboxy‐terminal repression domain is required for the development of normal flowers
Authors: Hiratsu Keiichiro , Ohta Masaru , Matsui Kyoko , Ohme-Takagi Masaru
Published: July 2002

SUPERMAN was identified as a putative regulator of transcription that acts in floral development, but its function remains to be clarified. We demonstrate here that SUPERMAN is an active repressor whose repression domain is located in the carboxy‐terminal region. Ectopic expression of SUPERMAN that lacked the repression domain resulted in a phenotype similar to that of superman mutants, demonstrating that the repression activity of SUPERMAN is essential for the development of normal flowers. Constitutive expression of SUPERMAN resulted in a severe dwarfism but did not affect cell size, indicating that SUPERMAN might regulate genes that are involved in cell division.
Expression of active, human lysyl oxidase in Escherichia coli
Authors: Ouzzine M. , Boyd A. , Hulmes D.J.S.
Published: July 2002

Lysyl oxidase (LO) is a copper amine oxidase of the extracellular matrix which initiates covalent cross‐linking in collagens and elastin. Human LO was expressed in Escherichia coli. At 37°C, large amounts of protein were obtained, but in the form of insoluble aggregates. Lowering the growth temperature, and reducing the amount of inducer, resulted in the production of soluble LO, which was active on a [3H]lysine‐labeled elastin substrate. LO was also targeted to the periplasm as a fusion protein with the pelb signal peptide. The periplasmic enzyme was soluble, active and inhibited by β‐aminopropionitrile. Production of the carbonyl co‐factor is therefore not a limitation in the expression of active LO in bacteria.
Estrogen receptor mRNA in mineralized tissues of rainbow trout: calcium mobilization by estrogen
Authors: Armour Kenneth J. , Lehane David B. , Pakdel Farzad , Valotaire Yves , Russell R.Graham G. , Henderson Ian W.
Published: July 2002

RT‐PCR was undertaken on total RNA extracts from bone and scales of the rainbow trout, Oncorhynchus mykiss. The rainbow trout estrogen receptor (ER)‐specific primers used amplified a single product of expected size from each tissue which, using Southern blotting, strongly hybridized with a 32P‐labelled rtER probe under stringent conditions. These data provide the first in vivo evidence of ER mRNA in bone and scale tissues of rainbow trout and suggest that the effects of estrogen observed in this study (increased bone mineral and decreased scale mineral contents, respectively) may be mediated directly through ER.
Deletion of the yeast homologue of the human gene associated with Friedreich's ataxia elicits iron accumulation in mitochondria
Authors: Foury Françoise , Cazzalini Ornella
Published: July 2002

Deletion of YDL120, the yeast homologue of the human gene responsible for Friedreich's ataxia, elicits decreased cellular respiration associated with decreased cytochrome c oxidase activity and, in certain nuclear backgrounds, mitochondrial DNA is lost. In the null mutants, the cellular growth is highly sensitive to oxidants, such as H2O2, iron and copper. However, only ferrous sulfate elicits loss of mitochondrial DNA. Mitochondria of the null mutants contain 10 times more iron than wild‐type. The neurodegeneration observed in Friedreich's ataxia can be well explained on the basis of a mitochondrial iron overload responsible for an increased production of highly toxic free radicals.
Avirulence gene D of Pseudomonas syringae pv. tomato may have undergone horizontal gene transfer
Authors: Hanekamp Theodor , Kobayashi Donald , Hayes Scott , Stayton Mark M.
Published: July 2002

Avirulence gene D (avrD) is carried on the B‐plasmid of the plant pathogen Pseudomonas syringae pv. tomato with plasmid‐borne avrD homologs widely distributed among the Pseudomonads. We now report sequences in the soft rot pathogen Erwinia carotovora that cross‐hybridize to avrD suggesting a conserved function beyond avirulence. Alternatively, avrD may have been transferred horizontally among species: (i) DNA linked to avrD shows evidence of class II transpositions and contains a novel IS3‐related insertion sequence, and (ii) short sequences linked to avrD are similar to pathogenicity genes from a variety of unrelated pathogens. We have also identified the gene cluster that controls B‐plasmid stability.
Purified HrpA of Pseudomonas syringae pv. tomato DC3000 reassembles into pili
Authors: Roine Elina , Saarinen Juhani , Kalkkinen Nisse , Romantschuk Martin
Published: July 2002

Pseudomonas syringae pv. tomato DC3000 produces Hrp pili under inducing in vitro conditions. A preparation of partially purified extracellular filaments contains HrpA, flagellin and some minor contaminants. HrpA was separated from the major contaminant, the flagellin, by gel filtration to a fraction containing HrpA as well as its three N‐terminally truncated forms. These were further separated by two steps of reversed phase chromatography. HrpA and its degradation products were each shown to reassemble into filament structures after denaturation and renaturation showing that HrpA alone is sufficient for formation of filament structures.
The Saccharomyces cerevisiae CCH1 gene is involved in calcium influx and mating
Authors: Fischer Marc , Schnell Norbert , Chattaway Jayne , Davies Paul , Dixon Graham , Sanders Dale
Published: July 2002

The yeast Saccharomyces cerevisiae gene CCH1 (ORF YGR217w) shows high homology with animal calcium channel α1‐subunit genes. Knock‐out mutants were constructed of Cch1 and of Mid1 which is known to mediate Ca2+ influx in response to the α‐mating pheromone. Cch1 is involved in calcium influx and the late stage of the mating process. The cch1 mutant sensitivity against the α‐mating pheromone can be reduced by the addition of extra calcium. The product of this gene is likely to interact with the MID1 gene product in Ca influx or its control.
Photoreceptor rhodopsin: structural and conformational study of its chromophore 11‐cis retinal in oriented membranes by deuterium solid state NMR
Authors: Gröbner Gerhard , Choi Gregory , Burnett Ian J , Glaubitz Clemens , Verdegem Peter J.E , Lugtenburg Johan , Watts Anthony
Published: July 2002

Rhodopsin is the retinal photoreceptor responsible for visual signal transduction. To determine the orientation and conformation of retinal within the binding pocket of this membrane bound receptor, an ab initio solid state 2H NMR approach was used. Bovine rhodopsin containing 11‐cis retinal, specifically deuterated at its methyl groups at the C19 or C20 position, was uniaxially oriented in DMPC bilayers. Integrity of the membranes and quality of alignment were monitored by 31P NMR. Analysis of the obtained 2H NMR spectra provided angles for the individual labelled chemical bond vectors leading to an overall picture for the three dimensional structure of the polyene chain of the chromophore in the protein binding pocket around the Schiff base attachment site.