bluuebunny/crossref_metadata_2025 huggingface dataset:
Fetal fibronectin as a screening test for premature delivery in multiple pregnancies
Authors: Tolino A. , Ronsini S. , Zullo F. , Pellicano M. , Regine V. , Nappi C.
Published: July 2002

Objective: To evaluate fetal fibronectin as a screening test for premature delivery in asymptomatic women with multiple pregnancies. Method: In the mid‐second trimester, the concentrations of fetal fibronectin in the cervical and vaginal secretions of 68 patients with multiple gestations were sampled weekly by monoclonal antibody immunoassay in order to predict preterm labor. Results: The results for the prediction of preterm labor differ according to whether we consider a single positive result (fetal fibronectin >50 ng/ml) as predictive of preterm labor or whether we only consider at least two consecutive positive results as predictive of preterm labor. The fetal fibronectin test had a sensitivity for preterm birth before 37 weeks of 90.9% and 86.6%, respectively, with a specificity of 68.5% vs. 78.9% and positive and negative values of 73.1% vs. 76.4% and 88.8% vs. 88.2%, respectively. Similar results were obtained for preterm birth before 34 weeks. Conclusion: In a condition such as multiple pregnancy which is already at risk for premature delivery the possibility of raising the specificity of the test with virtually no decrease in sensitivity guarantees better recognition of patients likely to develop premature labor. This possibility can be achieved simply by considering two positive consecutive samples as predictive of preterm labor.
Decidualized umbilical endometriosis
Authors: Skidmore R.A. , Woosley J.T. , Katz V.L.
Published: July 2002

A case of umbilical endometriosis is presented to highlight the challenges in its diagnosis. The etiology, clinical findings, histologic evaluation, prognosis and treatment options are discussed. While cyclic symptomatology may lend evidence to the diagnosis of umbilical endometriosis, history and clinical findings are often equivocal. Decidualization of umbilical endometriosis can be initially confused with a malignant process on histologic evaluation. The potential for malignant degeneration appears low. Surgical intervention is the treatment of choice.
Pregnancy with epilepsy — a retrospective analysis
Authors: Sawhney H. , Vasishta K. , Suri V. , Khunnu B. , Goel P. , Sawhney I.M.S.
Published: July 2002

Objectives: To study the course of epilepsy in pregnancy and to assess the perinatal outcome. Method: A retrospective analysis of 219 pregnant patients with epilepsy. The type of epilepsy, drug therapy and seizure frequency were documented. The perinatal outcome of 157 pregnancies with epilepsy was analyzed and compared with that of 471 normal gravidas of similar age and parity. Results: Generalized seizures occurred in 203 patients, partial seizures in 13 patients and complex partial seizures in three patients. One hundred fifty‐two patients (69.41%) were on monotherapy. Carbamazepine was the most common drug (56.58%) used. Ninety‐five patients (43.38%) had seizures in the current pregnancy, five of whom had status epilepticus. There was no maternal mortality in status epilepticus. There was no difference in perinatal outcome between the study and control groups. The incidence of congenital malformations was higher in the control group (5/476, 1.05%) than in the study group (1/160, 0.63%). The incidence of low‐birth‐weight babies was higher in the study group in patients with gestational seizures. Conclusion: The course of pregnancy and perinatal outcome was not altered by epilepsy. There was no increase in the incidence of congenital malformations with the use of monotherapy.
Key considerations for long-term studies with anxiolytics
Authors: Placchi M
Published: July 2002

Long-term (minimum six months) safety and efficacy studies with an anti-anxiety agent are aimed at identifying the emergence of any clinical effects not observed during shorter periods of treatment including the identification of late occurring adverse events, loss of therapeutic benefit, or post-treatment withdrawal effects. There is the need to establish diagnostic criteria for persistent anxiety for patients who truly benefit from long-term therapy. Risk factors predisposing to drug dependence should be assessed prior to continuous use. Ideally, long-term continuous studies should be blinded, comparative and placebo-controlled, with an adequate duration of the withdrawal phase. The treatment response of the elderly in comparison to the overall patient sample should be emphasised.
Conjugated linoleic acid induces lipid peroxidation in humans
Authors: Basu Samar , Smedman Annika , Vessby Bengt
Published: July 2002

Conjugated linoleic acid (CLA) is shown to have chemoprotective properties in various experimental cancer models. CLA is easily oxidised and it has been suggested that an increased lipid oxidation may contribute to the antitumorigenic effects. This report investigates the urinary levels of 8‐iso‐PGF2α, a major isoprostane and 15‐keto‐dihydro‐PGF2α, a major metabolite of PGF2α, as indicators of non‐enzymatic and enzymatic lipid peroxidation after dietary supplementation of CLA in healthy human subjects for 3 months. A significant increase of both 8‐iso‐PGF2α and 15‐keto‐dihydro‐PGF2α in urine was observed after 3 months of daily CLA intake (4.2 g/day) as compared to the control group (P<0.0001). Conjugated linoleic acid had no effect on the serum α‐tocopherol levels. However, γ‐tocopherol levels in the serum increased significantly (P=0.015) in the CLA‐treated group. Thus, CLA may induce both non‐enzymatic and enzymatic lipid peroxidation in vivo. Further studies of the mechanism behind, and the possible consequences of, the increased lipid peroxidation after CLA supplementation are urgently needed.
Pertussis toxin prevents presynaptic inhibition by kainate receptors of rat hippocampal [3H]GABA release
Authors: A. Cunha Rodrigo , Malva João O. , Ribeiro J.A.
Published: July 2002

Kainate receptors are ionotropic receptors, also reported to couple to Gi/Go proteins, increasing neuronal excitability through disinhibition of neuronal circuits. We directly tested in hippocampal synaptosomes if kainate receptor‐mediated inhibition of GABA release involved a metabotropic action. The kainate analogue, domoate (3 μM), inhibited by 24% [3H]GABA‐evoked release, an effect reduced by 76% in synaptosomes pre‐treated with pertussis toxin. Protein kinase C inhibition attenuated by 82% domoate‐induced inhibition of GABA release whereas protein kinase C activation did not change kainate receptor binding. Thus, domoate inhibition of GABA release recruits Gi/Go proteins and a protein kinase C pathway.
Membrane topology of the N‐terminus of the Escherichia coli FtsK division protein
Authors: Dorazi Robert , Dewar Susan J
Published: July 2002

The Escherichia coli FtsK protein targets the septum, is essential for cell division and may play a role in DNA partitioning. Computer modelling suggests that the first 180 amino acids of the protein are embedded in the cytoplasmic membrane by up to six transmembrane domains. We demonstrate, using gene fusions, that the N‐terminus contains four transmembrane helices that link two periplasmic domains. The first periplasmic domain contains an HEXXH amino acid sequence characteristic of zinc metalloproteases. We show by mutation analysis that the conserved glutamic acid of the HEXXH sequence is essential for FtsK function during septation.
Heteronuclear NMR studies of the specificity of the post‐translational modification of biotinyl domains by biotinyl protein ligase
Authors: Reche Pedro A. , Howard Mark J. , Broadhurst R.William , Perham Richard N.
Published: July 2002

The lipoyl domains of 2‐oxo acid dehydrogenase multienzyme complexes and the biotinyl domains of biotin‐dependent enzymes have homologous structures, but the target lysine residue in each domain is correctly selected for post‐translational modification by lipoyl protein ligase and biotinyl protein ligase, respectively. We have applied two‐dimensional heteronuclear NMR spectroscopy to investigate the interaction between the apo form of the biotinyl domain of the biotin carboxyl carrier protein of acetyl‐CoA carboxylase and the biotinyl protein ligase (BPL) from Escherichia coli. Heteronuclear multiple quantum coherence NMR spectra of the 15N‐labelled biotinyl domain were recorded in the presence and absence of the ligase and backbone amide 1H and 15N chemical shifts were evaluated. Small, but significant, changes in chemical shift were found in two regions, including the tight β‐turn that houses the lysine residue targetted for biotinylation, and the β‐strand 2 and the loop that precedes it in the domain. When compared with the three‐dimensional structure, sequence alignments of other biotinyl and lipoyl domains, and mutagenesis data, these results give a clear indication of how the biotinyl domain is both recognised by BPL and distinguished from the structurally related lipoyl domain to ensure correct post‐translational modification.
Binding of a de novo designed peptide to specific glycosaminoglycans
Authors: Jayaraman G , Wu C.W , Liu Y.J , Chien K.Y , Fang J.C , Lyu P.C
Published: July 2002

The binding of glycosaminoglycans to a synthetic peptide (SKAQKAQAKQAKQAQKAQKAQAKQAKQW–CONH2), consisting of a hybrid consensus heparin binding sequence, is studied using circular dichroism, fluorescence anisotropy and nuclear magnetic resonance techniques. The results unveil certain novel features, most importantly, the peptide binds preferentially to iduronic acid containing glycosaminoglycans and the dissociation constant for the peptide–heparin complex was found to be 30 nM. Interestingly, higher order intermolecular association(s)/aggregation was not observed, especially at saturating concentrations of the ligand. The helical structure of the peptide backbone, induced upon binding to a particular glycosaminoglycan is directly related to their binding affinity. In our opinion, studies on such unconventional hybrid peptide sequences containing low density basic amino acid residues would lead to the design of sequence specific glycosaminoglycan binding peptides.
Protein sorting in the Golgi apparatus: a consequence of maturation and triggered sorting
Authors: Weiss Matthias , Nilsson Tommy
Published: July 2002

To explain how resident proteins distribute in peak‐like patterns at various positions in the secretory pathway, Glick and co‐workers postulated that resident proteins comprise different populations (termed kin populations) and that these compete with each other for entering retrograde transport carriers [Glick et al. (1997) FEBS Lett. 414, 177–181]. Using modelling and computer simulation, they could demonstrate that differences in competitiveness sufficed to generate overlapping but distinct peak‐like steady state distributions of the different kin populations across the Golgi stack. In this study, we have tested the robustness of the competition model and find that over‐expression or changes in the number of kin populations affect their overall steady state distributions. To increase the robustness of the system, we have introduced a milieu‐induced trigger for recycling. This allows for a decrease in the coupling between kin populations permitting both over‐expression as well as changes in the number of kin populations. We have also extended the model to include a Golgi to endoplasmic reticulum (ER) recycling pathway and find that only a small amount of resident proteins may recycle at any time without upsetting their observed distributions in the Golgi stack. The biological relevance of a trigger‐induced sorting mechanism and ER recycling is discussed.
The N‐terminal domain of the Caulobacter crescentus CgtA protein does not function as a guanine nucleotide exchange factor
Authors: Lin Bin , Maddock Janine R
Published: July 2002

The Caulobacter crescentus GTP binding protein CgtA is a member of the Obg/GTP1 subfamily of monomeric GTP binding proteins. In vitro, CgtA displays moderate affinity for both GDP and GTP, and rapid exchange rate constants for either nucleotide. One possible explanation for the observed rapid guanine nucleotide exchange rates is that CgtA is a bimodal protein with a C‐terminal GTP binding domain and an N‐terminal guanine nucleotide exchange factor (GEF) domain. In this study we demonstrate that although the N‐terminus of CgtA is required for function in vivo, this domain plays no significant role in the guanine nucleotide binding, exchange or GTPase activity.
Antioxidant treatment induces transcription and expression of transforming growth factor β in cultured renal proximal tubular cells
Authors: Wolf Gunter , Hannken Tete , Schroeder Regine , Zahner Gunther , Ziyadeh Fuad N. , Stahl Rolf A.K.
Published: July 2002

Transforming growth factor β (TGF‐β) plays an important role in the development of tubulointerstitial fibrosis in chronic renal disease. We were interested whether interference with oxygen radicals may modulate TGF‐β expression. Unexpectedly, we discovered that diphenylene iodine (DIP), an inhibitor of NADP(H) oxidase, induces a robust increase in TGF‐β transcript expression in cultured mouse proximal tubular cells (MCT cells). A similar increase was seen with EUK‐8, a synthetic salen–manganese complex with high oxyradical scavenger activities. This induction of TGF‐β1 mRNA was paralleled by increasing protein expression. Transient transfection of MCT cells with a reporter construct in which murine TGF‐β1 enhancer/promoter elements were cloned in front of the luciferase gene, revealed that DIP, EUK‐8, and Tiron all stimulated transcription of the TGF‐β1 gene whereas exogenous H2O2 suppressed transcription. Antisense oligonucleotides against p22phox, but not sense oligonucleotides, also increased transcriptional activity of TGF‐β1. Mutagenesis of Sp1 binding sites in the mouse TGF‐β1 enhancer/promoter abolished the stimulatory effect of the antioxidants. Gel shift experiments revealed that DIP as well as EUK‐8 activated binding of nuclear proteins to Sp1 consensus sequence. Our data provide evidence that TGF‐β1 transcription is negatively regulated in MCT cells under basal conditions by NADP(H) oxidase‐mediated oxygen radicals. Thus, antioxidant therapy may increase local synthesis of TGF‐β1 in the tubulointerstitium.
Control of cell proliferation via transduction of sPLA2‐I activity and possible PPAR activation at the nuclear level
Authors: Specty O. , Pageaux J.F. , Dauça M. , Lagarde M. , Laugier C. , Fayard J.M.
Published: July 2002

Pancreatic phospholipase A2 (PLA2‐I) stimulates UIII cells proliferation, a rat uterine cell line, after binding to membrane receptors, internalization and translocation. Here, we demonstrate that during these steps of internalization, PLA2‐I retains its hydrolytic activity and thus could exert its proliferative effect via nuclear phospholipids hydrolysis. Since fatty acids and eicosanoids released by such activity are known to be ligands of PPAR, we study the expression of these nuclear receptors and demonstrate that, in the experimental conditions where PLA2‐I stimulates UIII cells proliferation, PLA2‐I also regulates PPAR expression indicating a possible mechanism of its proliferative effect.
Ser13‐phosphorylated PYY from porcine intestine with a potent biological activity
Authors: Chen Zheng-wang , Eriste Elo , Jonsson Andreas P. , Norberg Åke , Nepomuceno Diane , Lovenberg Timothy W. , Bergman Tomas , Efendic Suad , Jörnvall Hans , Sillard Rannar
Published: July 2002

We have isolated a posttranslationally modified form of peptide YY (PYY) from porcine intestine and shown by MALDI‐TOF and electrospray tandem mass spectrometry that it is phosphorylated at Ser13. Phospho‐PYY exhibits high affinity for binding to neuropeptide Y (NPY) receptors Y1, Y2 and Y5. The IC50 values with the Y1, Y2, and Y5 receptor subtypes were for NPY 2.4, 3.1, and 3.3 nM, for PYY 2.3, 0.94, and 3.2 nM, and for phospho‐PYY 4.6, 2.2, and 5.5 nM, respectively. Phospho‐PYY potently inhibits forskolin‐stimulated cAMP accumulation in SK‐N‐MC cells with an IC50 value of 0.5 nM compared to 0.15 nM for non‐phosphorylated PYY. The finding of phosphorylation of PYY is unusual among hormonal peptides, and emphasizes the importance of direct protein analysis of gene products.
Evolution and function of the neisserial dam‐replacing gene
Authors: Cantalupo Giuseppina , Bucci Cecilia , Salvatore Paola , Pagliarulo Caterina , Roberti Vera , Lavitola Alfredo , Bruni Carmelo Bruno , Alifano Pietro
Published: July 2002

Phase variation through slippage‐like mechanisms involving homopolymeric tracts depends in part on the absence of Dam‐methylase in several pathogenic isolates of Neisseria meningitidis. In Dam‐defective strains drg (dam‐replacing gene), flanked by pseudo‐transposable small repeated elements (SREs), replaced dam. We demonstrate that drg encodes a restriction endonuclease (NmeBII) that cleaves 5′‐GmeATC‐3′. drg is also present in 50% of Neisseria lactamica strains, but in most of them it is inactive because of the absence of an SRE‐providing promoter. This is associated with the presence of GATmeC, suggesting an alternative restriction‐modification system (RM) specific for 5′‐GATC‐3′, similar to Sau3AI‐RM of Staphylococcus aureus 3A, Lactococcus lactis KR2 and Listeria monocytogenes.
The predicted β12–β13 loop is important for inhibition of PP2Acα by the antitumor drug fostriecin
Authors: Evans David R.H , Simon Julian A
Published: July 2002

The potential anticancer agent fostriecin (FOS) is a potent inhibitor of the protein Ser/Thr phosphatases PP2A and PP4 and a weaker inhibitor of PP1. Random mutagenesis and automated screening in yeast identified residues in human PP2Acα important for inhibitory FOS binding. A C269S substitution in the predicted β12–β13 loop decreased the FOS sensitivity of intact cells and increased the IC50 of PP2Acα by 10‐fold in vitro. Changing PP2Acα Cys‐269 to phenylalanine, the equivalent residue in PP1, and the Y267G and G270D substitutions caused a similar effect. The results provide information relevant to the design of novel protein Ser/Thr phosphatase inhibitory drugs.
Transglutaminase‐mediated cross‐linking of α‐crystallin: structural and functional consequences
Authors: Shridas Preetha , Sharma Yogendra , Balasubramanian D.
Published: July 2002

Aggregation and covalent cross‐linking of the crystallins, the major structural proteins of the eye lens, increase light scattering by the lens leading to opacification and cataract. Disturbance of calcium homeostasis in the tissue is one of the factors implicated in cataractogenesis. Calcium‐activated transglutaminase (TG)‐catalyzed cross‐linking of some lens proteins has been reported earlier. We show here that α‐crystallin, a major structural protein in the lens and a member of the small heat shock protein family, is also a substrate for TG‐mediated cross‐linking, indicating the presence of donor Lys and acceptor Gln residues in the protein. Upon TG‐catalyzed dimerization, the secondary and tertiary structures of the protein are altered, and its surface hydrophobicity reduced. The chaperone‐like property of the protein, suspected to be one of its functions in situ, is substantially reduced upon such cross‐linking. These results, taken together with earlier ones on lens β‐crystallins and vimentin, suggest that TG‐mediated events might compromise lens function. Also, since α‐crystallin occurs not only in the lens but in other tissues as well, such TG‐catalyzed cross‐linking and the associated alterations in its structure and activity would be of general pathological interest.
Electrogenic reduction of the primary electron donor P700 by plastocyanin in photosystem I complexes
Authors: Mamedov Mahir D. , Mamedova Aygun A. , Chamorovsky Sergey K. , Semenov Alexey Yu.
Published: July 2002

An electrometric technique was used to investigate electron transfer between spinach plastocyanin (Pc) and photooxidized primary electron donor P700 in photosystem I (PS I) complexes from the cyanobacterium Synechocystis sp. PCC 6803. In the presence of Pc, the fast unresolvable kinetic phase of membrane potential generation related to electron transfer between P700 and the terminal iron–sulfur acceptor FB was followed by additional electrogenic phases in the microsecond and millisecond time scales, which contribute approximately 20% to the overall electrogenicity. These phases are attributed to the vectorial electron transfer from Pc to the protein‐embedded chlorophyll dimer P700+ within the PsaA/PsaB heterodimer. The observed rate constant of the millisecond kinetic phase exhibited a saturation profile at increasing Pc concentration, suggesting the formation of a transient complex between Pc and PS I with the dissociation constant K
d of about 80 μM. A small but detectable fast electrogenic phase was observed at high Pc concentration. The rate constant of this phase was independent of Pc concentration, indicating that it is related to a first‐order process.
Phosphorylation‐independent inhibition by intracellular cyclic nucleotides of brain inwardly rectifying K+ current expressed in Xenopus oocytes
Authors: Ito Hiroyuki , Tsuchimochi Hidetsugu , Tada Yoshihiko , Kurachi Yoshihisa
Published: July 2002

An inwardly rectifying K+ current, which was heterologously expressed in Xenopus oocytes, was inhibited by isoproterenol, a β‐adrenergic agonist. Poly(A)+ mRNA isolated from guinea‐pig brain was injected into oocytes 2–3 days before experiments. Isoproterenol inhibition of the K+ current was time‐ and voltage‐dependent: the inhibition became faster and more pronounced as the command voltage steps were applied to more negative potentials. This inhibition was prevented by propranolol. Dibutylyl cyclic (dB‐c) AMP could mimic the effect of isoproterenol, while injection of the catalytic subunit of cAMP‐dependent protein kinase into the oocytes did not affect the K+ current. Inhibitors of the protein kinases, WIPTIDE and H‐8, did not prevent the inhibition by dB‐cAMP. Furthermore, dB‐cGMP also inhibited the K+ current in a similar time‐ and voltage‐dependent manner. We propose that the phosphorylation‐independent action of cyclic nucleotides mediates β‐adrenergic inhibition of brain inwardly rectifying K+ channels expressed in Xenopus oocytes.
Enhanced catalytic activity of hexokinase by work‐induced mitochondrial binding in fast‐twitch muscle of rat
Authors: Parra Joan , Cusso Roser , Pette Dirk
Published: July 2002

© 1997 Federation of European Biochemical Societies.
In vitro cytotoxic effects of tumor necrosis factor‐α in human breast cancer cells may be associated with increased glucose consumption
Authors: Kaplan Ofer , Ruı́z-Cabello Jesús , Cohen Jack S
Published: July 2002

Tumor necrosis factor‐α inhibited growth of cultured MCF‐7 human breast cancer cells in a dose dependent manner. Tumor necrosis factor‐α also markedly increased glucose consumption, and its cytotoxicity was modified by glucose concentrations in the growth medium; higher glucose levels were associated with increased cell survival. However, when the cells were perfused in physiological conditions, very high levels of tumor necrosis factor‐α (200 ng/ml) in the perfusion solution had no inhibitory effects. Moreover, tumor necrosis factor‐α had no effects on 31P nuclear magnetic resonance spectra of the perfused cells. In the traditional growth inhibition assays, cells are incubated for several days with a drug, a situation where their metabolism is altered due to the depletion of nutrients, the accumulation of toxic waste materials and pH changes. Perfusion experiments are more relevant to in vivo conditions, and may be used for studying metabolic processes and the mechanisms of action of therapeutic agents.
Reconstitution of two isoforms of the human interleukin‐11 receptor and comparison of their functional properties
Authors: Lebeau Benoı̂t , Montero Julian Felix A , Wijdenes John , Müller-Newen Gerhard , Dahmen Heike , Chérel Michel , Heinrich Peter C , Brailly Hervé , Hallet Marie-Martine , Godard Anne , Minvielle Stéphane , Jacques Yannick
Published: July 2002

Long‐term stable Ba/F3 transfectants (B13Rα1 and B13Rα2) expressing two isoforms of the human IL‐11Rα receptor (α1 full length or α2 lacking the cytoplasmic domain) in combination with human gp130 were established. IL‐11Rα1 and IL‐11Rα2 were each expressed and detected as three bands upon Western blot analysis, with apparent molecular masses in agreement with those of the polypeptide backbone (47 and 44 kDa, respectively) with no, one or two N‐linked sugars. B13Rα1 and B13Rα2 bound IL‐11–thioredoxin with similar efficiencies and proliferated with superimposable dose–response curves to IL‐11, demonstrating that the intracellular domain of IL‐11Rα has no significant contribution on ligand binding and signaling. Analysis of a set of anti‐human gp130 mAbs confirmed the similar responsiveness of B13Rα1 and B13Rα2 transfectants.
The cytosolic aldehyde dehydrogenase gene (Aldh1) is developmentally expressed in Leydig cells
Authors: López-Fernández Luis A , del Mazo Jesús
Published: July 2002

Cytosolic aldehyde dehydrogenase, ALDH1, participates in the oxidation of different aldehydes including that of all‐trans retinal to retinoic acid. The accumulation of mouse Aldh1 transcripts is characterized by having different patterns in different tissues. This paper reports the greatest expression of Aldh1 in testis and liver. It was demonstrated that in testis, Aldh1 is specifically expressed in Leydig cells and is under developmental regulation. In vitro studies of cultured Leydig TM3 cells confirmed these results though such gene expression was found not to be mediated by LH regulation. Previous investigations have associated androgen receptors, and hence the androgen insensitivity syndrome in man, with the presence of ALDH1 in genital skin fibroblasts. However, this relationship was not established in a functional cell type, as is reported here for Leydig cells. These results could suggest a model for a molecular pathway from androgen receptor to retinoic acid biogenesis in Leydig cells via the mediation of ALDH.
Model of thec‐subunit oligomer in the membrane domain of F‐ATPases
Authors: Groth Georg , Walker John E.
Published: July 2002

A model is described of a dodecameric complex consisting of the integral membrane component subunitcof the H+‐transporting Fodomain ofEscherichia coliF‐ATPase. A high‐resolution partial structure of monomeric subunitcresulting from1H‐NMR studies[1]was used for constructing the model. The validity of the proposed arrangement of protomers in the dodecameric complex was tested by amino acid substitution analysis and chemical, biochemical and genetic data on subunitc.
Constitutive activation of the TSH receptor by spontaneous mutations affecting the N‐terminal extracellular domain
Authors: Duprez Laurence , Parma Jasmine , Costagliola Sabine , Hermans Jacques , Van Sande Jacqueline , Dumont Jacques E , Vassart Gilbert
Published: July 2002

Activating mutations of the TSH receptor gene have been found in toxic adenomas and hereditary toxic thyroid hyperplasia. Up to now, all mutations have been located in the serpentine portion of the receptor. We now describe two additional mutations affecting Ser‐281 (Ser‐281‐Thr and Ser‐281‐Asn) in the ectodomain of the receptor. After transfection in COS cells, both mutants displayed increased constitutive activity for cAMP generation despite expression at a lower level than the wild type. The mutants were responsive to TSH. The present results are compatible with a model in which the activity of the unliganded receptor is kept at a low level by an inhibitory interaction between the N‐terminal domain and the serpentine portion of the receptor.
Cell cycle specificity of Fas‐mediated apoptosis in WIL‐2 cells
Authors: Beletskaya Irina V , Nikonova Larisa V , Beletsky Igor P
Published: July 2002

Antibodies to Fas/APO1 receptor induce effective apoptosis in WIL‐2 cells of the human B‐lymphoid line. Quantitative assessment of the extent of the death in cells synchronized by thymidine block revealed a significant increase in their sensitivity to the cytocidal effect mediated by Fas/APO1 during the G1 phase of the cell cycle. Western analysis of the content of the p53 antigen in the cytoplasm and nuclei of the cells showed that the Fas/APO1‐induced death is accompanied by massive translocation of the p53 from the cytoplasm to the nucleus. These findings suggest that cell vulnerability to the Fas/APO1‐mediated apoptosis is subjected to regulation by cell cycle‐dependent mechanisms, one of which is probably the function of the p53 antigen.
Temperature dependence of the electrogenic reaction in the QB site of the Rhodobacter sphaeroides photosynthetic reaction center: the QA−QB→QAQB− transition
Authors: Gopta O.A. , Bloch D.A. , Cherepanov D.A. , Mulkidjanian A.Y.
Published: July 2002

The temperature dependencies for the kinetics and relative amplitudes of electrogenic reaction(s) coupled with the first reduction of the secondary quinone acceptor QB were measured with dark‐adapted chromatophores of Rhodobacter sphaeroides. The kinetics, while acceptably fitted by a single exponent at room temperature, clearly split into two components below 15°C (rise times, 25 μs and 300 μs at pH 7.0 and 10°C) with the slow phase ousting the fast one at pH>9.0. The activation energies of the fast and slow phases were estimated at pH 7.0 as <10 kJ/mol and 60–70 kJ/mol, respectively. To explain the kinetic heterogeneity of the QB→QB
− transition, we suggest two possible conformations for the neutral oxidized ubiquinone at the QB site: one with a hydrogen bond between the side chain carboxyl of Glu‐L212 and the methoxy oxygen at C3 of the QB ring (QB−H‐Glu centers) and the other one, without this bond (QB:Glu− centers). The fast phase is attributed to QA
−QB–H–Glu→QAQB–H–Glu transition, whereas the slow one to the QA
−QB:Glu−→QA
−QB–H–Glu→QAQB
−–H–Glu transition.
Drosophila alcohol dehydrogenase: evaluation of Ser139 site‐directed mutants
Authors: Cols Neus , Atrian Sı́lvia , Benach Jordi , Ladenstein Rudolf , Gonzàlez-Duarte Roser
Published: July 2002

Drosophila alcohol dehydrogenase (DADH) belongs to the large and highly heterogeneous (15–30% residue identity) short‐chain dehydrogenase/reductase family (SDR). It is the only reported member that oxidizes mainly ethanol and 2‐propanol among other alcohols. To confirm the role of Ser139 we constructed two site‐directed mutants, Ser139Ala and Ser139Cys, which show no enzymatic activity. Molecular replacement and data from crystallographically refined 3D structures confirm the position of Ser139, whose hydroxyl group faces the cleft of the presumed catalytic pocket, very close to Tyr152 and Lys156. Thus, consistent with the constitution of the catalytic triad of other SDR, our results suggest that Ser139 of DADH is directly involved in the catalytic reaction.
MPM‐2 epitope sequence is not sufficient for recognition and phosphorylation by ME kinase‐H
Authors: Che Shaoli , Weil Michael M , Nelman-Gonzalez Mayra , Ashorn Cheryl L , Kuang Jian
Published: July 2002

Monoclonal antibody MPM‐2 recognizes a large family of mitotic phosphoproteins in a phosphorylation‐dependent manner. The antigenic phosphoepitope, designated the MPM‐2 epitope, putatively consists of hydrophobic residue‐Thr/Ser‐Pro‐hydrophobic residue‐uncharged/basic residue. In this study, we addressed whether this sequence motif contains all the information necessary for recognition and phosphorylation by the kinase that phosphorylates most MPM‐2 antigens. A fusion protein between glutathione S‐transferase and a 19‐residue peptide that contained two representative MPM‐2 epitope sequences overlapping with two potential MAP kinase phosphorylation sites was constructed. Both the MPM‐2 epitope sequences in the fusion protein (GST‐MPM2) were phosphorylated by Xenopus egg extract, making the fusion protein MPM‐2 reactive. However, while MAP kinase phosphorylated both the MPM‐2 epitope sequences, neither ME kinase‐H, a good candidate for a major MPM‐2 epitope kinase, nor mitotic cdc2 kinase, which is known to phosphorylate certain MPM‐2 antigens in vitro, phosphorylated GST‐MPM2 to any significant extent. Furthermore, depletion of MAP kinase activity removed most, if not all, of the GST‐MPM2 phosphorylating activity from crude Xenopus egg extracts. These results suggest that additional or different structural information than that provided by the deduced MPM‐2 epitope sequence is required for recognition and phosphorylation by ME kinase‐H or other major MPM‐2 epitope kinases. They also offer a valid explanation for selective phosphorylation of certain MPM‐2 antigens by MAP kinase as well as selective recognition of certain phosphorylated MAP kinase substrates by MPM‐2.
Matrilin‐3 from chicken cartilage
Authors: Belluoccio Daniele , Trueb Beat
Published: July 2002

By subtractive cDNA cloning we have identified a novel constituent of chicken cartilage termed matrilin‐3. This constituent is encoded by a mRNA of 2.2 kbp whose expression is restricted to cartilaginous tissues. The predicted protein is composed of 452 amino acids with a molecular mass of 49 kDa. It contains a single von Willebrand factor A‐like domain, four epidermal growth factor‐like repeats and an α‐helical region which may induce the formation of oligomers via a coiled‐coil. The primary structure is similar to that of matrilin‐1 which is also expressed in a cartilage‐specific manner. This similarity suggests that the genes for the two proteins may have evolved from a common ancestor by gene duplication.
Functional assessment of the yeast Rvs161 and Rvs167 protein domains
Authors: Sivadon Pierre , Crouzet Marc , Aigle Michel
Published: July 2002

Mutations in RVS161 and RVS167 yeast genes induce identical phenotypes associated to actin cytoskeleton disorders. The whole Rvs161 protein is similar to the amino‐terminal part of Rvs167p, thus defining a RVS domain. In addition to this domain, Rvs167p contains a central glycine‐proline‐alanine rich domain and a SH3 domain. To assess the function of these different domains we have expressed recombinant Rvs proteins in rvs mutant strains. Phenotype analysis has shown that the RVS and SH3 domains are necessary for phenotypical complementation, whereas the GPA domain is not. Moreover, we have demonstrated that the RVS domains from Rvs161p and Rvs167p have distinct roles, and that the SH3 domain needs the specific RVS domain of Rvs167p to function. These results suggest that Rvs161p and Rvs167p play distinct roles, while acting together in a common function.
Gα16 protein expression is up‐ and down‐regulated following T‐cell activation: disruption of this regulation impairs activation‐induced cell responses
Authors: Lippert Eric , Baltensperger Kurt , Jacques Yannick , Hermouet Sylvie
Published: July 2002

The role of heterotrimeric G proteins in T‐cell activation is poorly understood. Here we show that in normal, mature human T‐cells, expression of Gα16, the 43 kDa α subunit of G16, varies widely, depending on T‐cell activation status. Quiescent blood lymphocytes strongly up‐regulate Gα16 after Leuco A stimulation: protein expression of Gα16 is maximal at day 4, then decreases. Consistently, in human T‐cell clones, expression of Gα16 is high in the first week following activation and decreases rapidly within the second week. In addition, permanent disruption of regulated Gα16 expression in Jurkat T‐cells by stable overexpression of 43 kDa Gα16 inhibited Leuco A‐induced interleukin‐2 production, CD69 up‐regulation and cell apoptosis (by 58%, 46% and 74%, respectively), suggesting that coordinate regulation of Gα16 expression is necessary for optimal activation‐induced T‐cell responses, and that Gα16 proteins may be involved in the negative regulation of TCR signalling.
Biotin synthesis in higher plants: purification and characterization of bioB gene product equivalent from Arabidopsis thaliana overexpressed in Escherichia coli and its subcellular localization in pea leaf cells
Authors: Baldet Pierre , Alban Claude , Douce Roland
Published: July 2002

Biotin synthase catalyses the final step in the biotin biosynthetic pathway and is encoded by the bioB gene in Escherichia coli. To investigate the conversion of dethiobiotin to biotin in the plant kingdom, the cDNA encoding the bioB gene product equivalent from Arabidopsis thaliana was used to construct an E. coli overexpression strain. The purified A. thaliana bioB gene product is a homodimer (100 kDa) with a reddish color and has an absorbance spectrum characteristic of protein with [2Fe‐2S] clusters. Its intracellular compartmentation in pea leaves discloses a unique polypeptide of 39 kDa within the matrix of mitochondria.
Purification and characterization of a group of five novel peptide serine protease inhibitors from ovaries of the desert locust, Schistocerca gregaria
Authors: Hamdaoui Ahmed , Wataleb Said , Devreese Bart , Chiou Shean-Jaw , Vanden Broeck Jozef , Van Beeumen Jozef , De Loof Arnold , Schoofs Liliane
Published: July 2002

The ovary of the desert locust, Schistocerca gregaria, contains multiple inhibitors of serine proteases. Five serine protease inhibitors, designated SGPI‐1–5 (Schistocerca gregaria protease inhibitors) were purified from methanolic extracts of mature ovaries and analyzed by mass spectrometry and amino acid sequencing. The revealed primary structures display amino acid similarities and are related to the serine protease inhibitors identified in the hemolymph of Locusta migratoria. All inhibitors show an in vitro inhibiting activity towards α‐chymotrypsin. In addition, SGPI‐1 displays in vitro inhibiting activity towards trypsin, and SGPI‐2 is a potent pancreatic elastase inhibitor. Differences in inhibitory specificities towards the locust endogenous serine proteases can be readily attributed to the amino acid sequence within the active region and also to amino acid residues beyond the P1‐P′1 bond. A difference in one or two amino acid residues around the reactive sites results in considerable alteration of the inhibitory specificity. The temporal and spatial distribution of SGPI‐1–5 was studied by RP‐HPLC analysis. All inhibitors occur in hemolymph, ovaries, testes and fat body of adults but are absent in the gut. They are also present in larval hemolymph and fat body. An antibody raised against SGPI‐2 shows positive immunostaining in the ovarian follicle cells.
Homology between a human apoptosis specific protein and the product of APG5, a gene involved in autophagy in yeast
Authors: Hammond Ester M , Brunet Clare L , Johnson Gerald D , Parkhill Julian , Milner Anne E , Brady Ged , Gregory Christopher D , Grand Roger J.A
Published: July 2002

Apoptosis specific proteins (ASP) are expressed in the cytoplasm of cultured mammalian cells of various lineages following induction of apoptosis. The cDNA encoding ASP has been cloned from a human expression library and has significant homology to the Saccharomyces cerevisiae APG5 gene which is essential for yeast autophagy. The ASP gene, known as hAPG5, can be transcribed to give mRNAs of 3.3 kbp, 2.5 kbp and 1.8 kbp which are present at comparable levels in viable and apoptotic cells, demonstrating that protein expression must be regulated at the translational level. These data indicate a possible relationship between apoptosis and autophagy and suggest evolutionary conservation in mammalian apoptosis of a degradative process present in yeast.
Profile of protein kinase C isozymes and their possible role in mammalian egg activation
Authors: Raz Tamar , Eliyahu Efrat , Yesodi Vered , Shalgi Ruth
Published: July 2002

Western blot analysis was used to investigate protein kinase C (PKC) profile of rat eggs. The presence of eight PKC isozymes was demonstrated: conventional PKC α, β and γ; novel PKC δ, ϵ and μ; atypical PKC ζ and λ. PKC α was detected by RT‐PCR as well. PKC translocation from the cytosol to the plasma membrane served as a marker for enzyme activation. Immunofluorescence confocal microscopy demonstrated a relatively uniform distribution of PKC α, βI, and βII throughout the cytosol of metaphase II arrested eggs. PKC accumulation at the plasma membrane was detected 5 min after exposure to 12‐O‐tetradecanoyl phorbol‐13‐acetate and increased with time, thus demonstrating activation of these PKCs.
Cloning of a shrimp (Metapenaeus ensis) cDNA encoding a nuclear receptor superfamily member: an insect homologue of E75 gene1
Authors: Chan Siu-Ming
Published: July 2002

Degenerate primers were derived from the amino acid sequence in the DNA binding domain of the Drosophila ecdysone receptor (DmEcR). Several partial cDNAs were amplified from the shrimp epidermis by reverse transcription polymerase chain reaction (RT‐PCR). One of these fragments shows the highest amino acid sequence homology to the insect ecdysone inducible gene E75. This partial cDNA was used as a probe to screen the swimming leg cDNA library of the shrimp, Metapenaeus ensis. A 3.6 kb cDNA clone was obtained. The longest open reading frame of this cDNA consists of 606 amino acids and its deduced amino acid sequence has all five domains typical of a nuclear receptor. The putative polyadenylation signal is located at about 400 bp 3′ to the stop signal. The deduced amino acid sequence of this cDNA shows the highest identity to that of the E75A reported in Manduca sexta, Galleria melonella, Drosophila melanogaster, and Choristoneura fumiferana. Based on the amino acid sequence comparison, the shrimp nuclear receptor is considered the insect homologue of E75A. Northern blot analysis shows that the shrimp E75 is expressed in the epidermis, eyestalk and the nerve cord of the pre‐molt shrimp. Moreover, E75 transcripts can be detected in the epidermal tissues of early pre‐molt shrimp by in situ hybridization. To determine whether the shrimp could also express other E75s like the insects, 5′ end RACE and RT‐PCR were performed on epidermal cDNA of a single shrimp. Subcloning and DNA sequence determination of the PCR products confirmed the presence of two other forms of E75 (tentatively called E75C and E75D) in shrimp. By RT‐PCR, different levels of E75 expression can be detected in the epidermis, nerve cord and the eyestalk of early pre‐molt shrimp. In addition to the different levels of expression of the shrimp E75s in the epidermis, the pattern of their expression is also different during the molting cycle. This is the first report on the cloning of a shrimp nuclear receptor superfamily member.
Inhibition of the double‐stranded RNA‐dependent protein kinase PKR by mammalian ribosomes
Authors: Raine Daniel A , Jeffrey Ian W , Clemens Michael J
Published: July 2002

Previous evidence has shown that the majority of the interferon‐inducible, double‐stranded RNA‐dependent protein kinase PKR is associated with ribosomes in vivo. Here we show that ribosomes are inhibitory for PKR activity since they compete with dsRNA for binding to PKR, inhibit the activation of the protein kinase by dsRNA, and prevent the phosphorylation of the PKR substrate eIF2α. We suggest that ribosomes constitute a reservoir of inactive PKR and that the protein kinase must be displaced from the ribosome by dsRNA in order to become activated.
Immediate early response of the p62 gene encoding a non‐proteasomal multiubiquitin chain binding protein
Authors: Lee Young Han , Ko Jesang , Joung Insil , Kim Jung-Hye , Shin Jaekyoon
Published: July 2002

p62 is a cytoplasmic ubiquitin chain binding protein. Upon a variety of extracellular signals, both transcript and protein levels of p62 were rapidly increased. These signals include phorbol 12‐myristate 13‐acetate (PMA) and calcium ionomycin for peripheral blood mononuclear cells, serum or PDGF for serum‐starved NIH3T3 cells, IL‐3 for the G1 arrested pre‐B cell line Ba/F3, and PMA for a human promyelocyte line U937. The elevation of p62 transcript level is due to temporal stabilization of mRNA and rapid activation of the p62 gene. Cycloheximide‐induced enhanced transcription suggests the immediate early response of the p62 gene. The rapid induction of p62 indicates the presence of a novel ubiquitination‐mediated regulatory mechanism during cell proliferation and differentiation.
Kinetic and spectroscopic characterization of an intermediate peroxynitrite complex in the nitrogen monoxide induced oxidation of oxyhemoglobin
Authors: Herold Susanna
Published: July 2002

Stopped‐flow spectroscopy studies of the nitrogen monoxide mediated oxidation of oxyhemoglobin in the pH range 5–10.5 show that an intermediate can be characterized at alkaline pH. The rate of decay of this species to methemoglobin increases significantly with decreasing pH such that it does not accumulate in quantities large enough to be observed under neutral or acidic conditions. Kinetic and spectroscopic properties of this intermediate support its assignment as a methemoglobin peroxynitrite complex.
Crystal structure of rabbit muscle creatine kinase1
Authors: Mohana Rao J.K , Bujacz Grzegorz , Wlodawer Alexander
Published: July 2002

The crystal structure of rabbit muscle creatine kinase, solved at 2.35 Å resolution by X‐ray diffraction methods, clearly identified the active site with bound sulfates surrounded by a constellation of arginine residues. The putative binding site of creatine, which is occupied by a sulfate group in this analysis, has been tentatively identified. The dimeric interface of the enzyme is held together by a small number of hydrogen bonds.
Platelet high affinity low density lipoprotein binding and import of lipoprotein derived phospholipids
Authors: Dobner Petra , Koller Elisabeth , Engelmann Bernd
Published: July 2002

The binding of low density lipoprotein (LDL) to the platelet cell membrane could facilitate the transfer of phospholipids from LDL to the platelets. A polyclonal antibody against the platelet glycoproteins IIb/IIIa inhibited the high affinity binding of 125I‐LDL by up to 80%. The transfer of pyrene (py)‐labeled sphingomyelin (SM), phosphatidylcholine and phosphatidylethanolamine from LDL to the platelets was unaffected by the antibody. The lectin wheat germ agglutinin (WGA) reduced the binding of 125I‐LDL to the platelets by approximately 80%. In contrast, the lectin stimulated the transfer of SM from LDL into the platelets by about three‐fold. WGA also specifically augmented the transfer of py‐SM between lipid vesicles and the platelets, the stimulation being abolished in the presence of N‐acetylglucosamine. Dextran sulfate (DS) increased the specific binding of 125I‐LDL to the platelets by up to 2.8‐fold. On the other hand, the import of LDL‐derived py‐phospholipids was unaffected by DS. Together, the results indicate that the phospholipid transfer from LDL to the platelets is independent of the high affinity LDL binding to the platelets and is specifically stimulated by WGA. Thus, the interactions of platelets with LDL phospholipids differ markedly from those with the apoprotein components of the lipoproteins.
Inhibition of nucleoside diphosphate kinase in rat liver mitochondria by added 3′‐azido‐3′‐deoxythymidine
Authors: Valenti Daniela , Barile Maria , Quagliariello Ernesto , Passarella Salvatore
Published: July 2002

The effect of 3′‐azido‐3′‐deoxythymidine on nucleoside diphosphate kinase of isolated rat liver mitochondria has been studied. This is done by monitoring the increase in the rate of oxygen uptake by nucleoside diphosphate (TDP, UDP, CDP or GDP) addition to mitochondria in state 4. It is shown that 3′‐azido‐3′‐deoxythymidine inhibits the mitochondrial nucleoside diphosphate kinase in a competitive manner, with a K
i value of about 10 μM as measured for each tested nucleoside diphosphate. It is also shown that high concentrations of GDP prevent 3′‐azido‐3′‐deoxythymidine inhibition of the nucleoside diphosphate kinase.