bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptional‐ and post‐transcriptional‐dependent regulation of glutathione S‐transferase expression in rat hepatocytes as a function of culture conditions
Authors: Vandenberghe Yves , Tee Lisa , Rogiers Vera , Yeoh George
Published: July 2002

Transcriptional activity of the glutathione S‐transferase (GST) α (subunits 1 and 2), μ (subunits 3 and 4) and π (subunit 7) gene families has been analyzed using the nuclear ‘run‐on’ technique on adult rat hepatocytes maintained for 4 days in conventional culture and for 4 and 12 days in co‐culture with rat liver epithelial cells. Several medium conditions are included in this study, namely with or without fetal calf serum and with nicotinamide or dimethylsulphoxide. Hepatocytes co‐cultured for 4 days maintain approximately 30–70% of the α gene family transcriptional activity, whatever the medium conditions, when compared to freshly isolated hepatocytes. A marked decrease is observed after 12 days of co‐culture or when hepatocytes are maintained in conventional culture. The transcriptional activity of the μ gene family is maintained at 40–160% when hepatocytes are cultured with or without fetal calf serum, and is inducible by nicotinamide (approximately 4‐fold) and dimethylsulphoxide (approximately 2‐fold) in conventional culture and/or in co‐culture. In contrast to freshly isolated hepatocytes, GST π gene transcriptional activity is observed in conventional and co‐cultured hepatocytes, irrespective of the medium conditions. Dimethylsulphoxide treatment however, represses the expression of GST 7 in vitro. These results demonstrate that the expression of GST α, μ and π genes in conventional and co‐cultured rat hepatocytes is controlled primarily at the level of transcription. It cannot be excluded, however, that dimethylsulphoxide stabilizes the GST mRNA levels in vitro.
The effect of hydroxyl radical on the antigenicity of native DNA
Authors: Alam Khurshid , Ali Asif , Ali Rashid
Published: July 2002

Hydroxyl free‐radical mediated in vitro modification of native calf thymus DNA showed single‐strand breaks, decrease in melting temperature and structural alteration of purine and pyrimidine bases. Experimentally induced antibodies against modified DNA exhibited polyspecificity. Native DNA, RNA and synthetic polynucleotides in B‐conformation were found to be an effective inhibitor of induced antibody‐immunogen interaction. Naturally occurring human anti‐DNA autoantibodies showed enhanced recognition of modified DNA as compared to native polymer. IgG isolated from anti‐DNA antibody‐positve SLE sera by Protein A‐Sepharose 4B chromatography were affinity purified on nDNA‐(polylysyl‐Sepharose 4B) matrix. In competitive binding experiments, the affinity‐isolated IgG showed almost equal recognition of both native DNA and hydroxyl radical modified DNA. The possible role of hydroxyl radicals in the etiopathogenesis of SLE has been discussed.
Primary structure and functional expression of mouse pituitary and human brain corticotrophin releasing factor receptors
Authors: Vita Natalio , Laurent Patrick , Lefort Sylvie , Chalon Pascale , Lelias Jean-Michel , Kaghad Mourad , Le Fur Gérard , Caput Daniel , Ferrara Pascual
Published: July 2002

Corticotrophin‐releasing factor (CRF) is the principal hypothalamic factor governing the pituitary‐adrenal axis, but the wide extra‐pituitary distribution of CRF and its receptors suggest a major role for this neuropeptide in the integration of the overall physiological and behavioral responses of an organism to stress. We have cloned a CRF receptor complementary DNA (cDNA) by expression in COS‐7 cells of a cDNA library from the AtT20 mouse pituitary tumour cell line. The cloned mouse cDNA was then used as a probe to isolate a human CRF receptor cDNA from a human brain cDNA library. The mouse and human cDNAs both encode 415 amino acid proteins that are 97% identical, containing seven putative transmembrane domains characteristic of G protein‐coupled receptors. The CRF receptor shows homology with the receptors for growth hormone‐releasing factor, vasoactive intestinal peptide, secretin, parathyroid hormone, and calcitonin. COS‐7 cells transfected with the mouse CRF receptor cDNA bind radiolabelled ovine CRF with high affinity and respond specifically to CRF by accumulation of intracellular cAMP. A 2.7 kb mRNA coding for the CRF receptor could be detected in AtT20 cells and human cortex tissue. PCR analysis also detected the receptor transcript in human pituitary, brainstem, and testis.
The relationships between transketolase, yeast pyruvate decarboxylase and pyruvate dehydrogenase of the pyruvate dehydrogenase complex
Authors: Robinson Brian H. , Chun Kathy
Published: July 2002

The amino acid sequences of four thiamine pyrophosphate‐requiring enzymes were aligned with the published amino acid sequence of the transketolase of Hansenula polymorpha. Sequences of the combined α and β subunits of the E1 enzyme of the pyruvate dehydrogenase complexes of Homo sapiens and Bacillus stearothermophilus aligned well with the transketolase while the E, of the pyruvate dehydrogenase complex of Escherichia coli aligned easily provided a non‐aligning segment of 77 amino acids was omitted. The non‐acetylating pyruvate decarboxylase of Saccharomyces cerevisiae could only be aligned if the sequence was cut in two with the C‐terminus corresponding to the N‐terminus of the other TPP‐dependent enzymes. Using the published 2.5 Å resolution of the X‐ray crystal structure of rSaccharomyces cerevisiae transketolase as a template we show that a hydrophobic region of the β‐subunit of the PDH E1 αβ enzymes likely contains a binding site for the thiazolium ring of TPP and key motifs are retained in common by all the TPP‐dependent enzymes considered, which are essential for catalysis.
Differentiation of HL60 promyelocytic cells is promoted by a ‘differentiation enhancing factor’ produced by erythroleukemia cells
Authors: Sparatore B. , Passalacqua M. , Patrone M. , Pessino A. , Melloni E. , Pontremoli S.
Published: July 2002

A differentiation enhancing factor isolated from murine erythroleukemia cells is also a potent enhancer of the differentiation of HL60 human promyelocytic leukemia cells, induced by retinoic acid and by phorbol ester. This stimulating effect is the result of a large increase in the sensitivity of HL60 cells for retinoic acid and for phorbol 12‐myristate 13‐acetate (20‐fold and 40‐fold, respectively). Accelerated differentiation induced by the protein factor, and monitored by the appearance of marker enzymes, is accompanied by a large increase in the fluctuation of the levels of protein kinase C (PKC) isozymes in HL60 cells. These results provide further support for the role of this new protein factor in cell differentiation and indicate that other cell types are susceptible to its biological effect.
Photochemical labeling of membrane‐associated and channel‐forming domains of proteins directed by energy transfer
Authors: Peng Ling , Alcaraz Marie-Lyne , Klotz Philippe , Kotzyba-Hibert Florence , Goeldner Maurice
Published: July 2002

Singlet—singlet energy transfer reactions from excited tryptophan residues to photoactivatable probes possessing a suitable chromophore, generate reactive species in the vicinity of the protein, leading to its covalent labeling. This delayed labeling process can be used to map the membrane‐surrounded regions of proteins with improved efficiency when it is applied with appropriate photoactivatable phospholipids. The same principle could also be applied to the labeling of channel‐forming transmembrane domains of ion channels, provided that suitable photoactivatable permeant ions were available. Both applications will be discussed with regard to their potential and feasibility.
Membrane bending energy concept of vesicle‐ and cell‐shapes and shape‐transitions
Authors: Sackmann E.
Published: July 2002

The main objective of this lecture is to discuss the role of lipid‐bilayer elasticity (1) for the self‐organization of lipid/protein‐bilayers (2) for the stabilization of domain structures and shapes of cell membranes and (3) for the control of shape transitions (e.g. bud‐ and pit‐formation) and shape instabilities (vesicle fission). It is demonstrated that many complex shape transitions of cell membranes can be mimicked by single lipid bilayer vesicles by simply varying the area‐to‐volume ratio or by chemically induced bending moments suggesting that these processes are governed by the universal minimum bending energy concept of closed shells composed of stratified membranes. The essential role of the coupling between curvature and phase separation in mixed membranes for the formation and stabilization of local pits and buds or the fission of budded vesicles is demonstrated. Finally, we discuss the consequences of the pronounced thermally excited bending undulations of the hyperelastic membranes for the membrane tension, the material exchange at membrane surfaces and the control of the adhesion of vesicles (or cells) on solid substrates.
cDNA Cloning of an orphan opiate receptor gene family member and its splice variant
Authors: Wang Jia Bei , Johnson Peter S. , Imai Yasuo , Persico Antonio M. , Ozenberger Bradley A. , Eppler C.Mark , Uhl George R.
Published: July 2002

Radioligand binding and cDNA homology studies have suggested the existence of opiate receptors distinct from the recently‐cloned, μ, δ and κ receptors. XOR1S, a rat brain cDNA whose predicted translation product displays 67–72% homology with those encoded by μ1, δ1 and κ1 opiate receptor cDNAs, was constructed from two partial cDNAs identified through cDNA homology approaches. A longer XOR1L variant of this cDNA was also identified by polymerase chain reaction studies using genomic DNA and cDNA from brain and peripheral tissues. XOR1 mRNA is most highly expressed in hypothalamus. COS cell expression of both clones confers neither robust binding of opiate ligands nor reproducible opiate inhibition of forskolin‐stimulated adenylate cyclase. These studies identify an orphan clone that helps to define features of the opiate receptor gene family, including apparent differential splicing and expression in peripheral tissues.
Intense oxidative DNA damage promoted by l‐DOPA and its metabolites implications for neurodegenerative disease
Authors: Spencer Jeremy P.E. , Jenner Andrew , Aruoma Okezie I. , Evans Patricia J. , Kaur Harparkash , Dexter David T. , Jenner Peter , Lees Andrew J. , Marsden David C. , Halliwell Barry
Published: July 2002

Oxidative DNA damage can cause mutation and cell death. We show that l‐DOPA, dopamine and 3‐O‐methyl‐DOPA cause extensive oxidative DNA damage in the presence of H2O2 and traces of copper ions. 8‐Hydroxyguanine is the major product. Iron ions were much less effective and manganese ions did not catalyse DNA damage. We propose that copper ion release, in the presence of l‐DOPA and its metabolites, may be an important mechanism of neurotoxicity, e.g. in Parkinson's disease and amyotrophic lateral sclerosis.
Three‐dimensional crystal structure of recombinant erabutoxin a at 2.0 Å resolution
Authors: Arnoux Bernadette , Ménez Renée , Drevet Pascal , Boulain Jean-Claude , Ducruix Arnaud , Ménez André
Published: July 2002

Recombinant erabutoxin a (Ear) has been crystallized by vapour diffusion in hanging drops. The crystals belong to space group P212121 with cell dimensions a = 55.8 Å, b = 53.4 Å, c = 40.8 Å. Diffraction data have been recorded on a FAST detector up to 2.0 Å. The atomic crystal structure of Ear has been determined by initial refinement of the structure of the isotoxin erabutoxin b (Eb) the crystals of which were grown under identical conditions. The R‐factor was 23% at 2.0 Å resolution. The secondary and tertiary structures of Ear are shown to be identical with that of wild‐type Eb, within the experimental error.
Chimeric alpha‐beta oligonucleotides as antisense inhibitors of reverse transcription
Authors: Boiziau Claudine , Debart Françoise , Rayner Bernard , Imbach Jean-Louis , Toulme Jean-Jacques
Published: July 2002

Alpha‐beta chimeric 17‐mer oligodeoxyribonucleotides containing either 5, 10 or 15 beta nucleotides were synthesized. The stability of the RNA/chimera hybrids was only slightly affected by the alpha stretch and by the alpha‐beta link, as was the affinity of the Moloney Murine Leukemia Virus reverse transcriptase for the duplexes. All chimeras inhibited in vitro cDNA synthesis in a cell‐free system to various extent, via the degradation of the RNA target by RNase H.
The Grb2 adaptor
Authors: Chardin Pierre , Cussac Didier , Maignan Sébastien , Ducruix Arnaud
Published: July 2002

Grb2 is an ‘adaptor’ protein made of one SH2 and two SH3 domains. The SH3 domains bind to prolinerich motifs in the C‐terminal part of the ras exchange factor Sos. Binding of the Grb2 SH2 domain to phosphotyrosine motifs on receptors, or other adaptor proteins such as Shc, recruits this Grb2/Sos complex at the plasma membrane where Sos stimulates nucleotide exchange on ras, then ras activates raf and leads to MAP kinase activation. The structure of Grb2, the precise motifs recognised by its SH2 and SH3 domains, the way Grb2 performs its function, a possible regulation of its association with Sos, and its ability to complex with other proteins in vivo, are discussed.
Proteolytic processing of nuclear factor κB by calpain in vitro
Authors: Liu Zhen-Qiu , Kunimatsu Mitoshi , Yang Jian-Ping , Ozaki Yasuhiko , Sasaki Makoto , Okamoto Takashi
Published: July 2002

Nuclear factor KB (NF‐κB) is a transcription factor that is critical for the inducible expression of multiple cellular and viral genes. Using the electrophoretic mobility shift assay, we demonstrated that DNA binding activity of NF‐κB was abolished by proteolysis with μ‐ and m‐calpains in vitro. The proteolysis of NF‐κB by calpains and hence the abolition of its DNA binding was prevented by calpastatin, calpain inhibitor I and proteasome inhibitor. We also provided evidence that calpains degrade the Cterminal domain of NF‐κB by Western blot using anti‐NF‐κB (p65) C‐terminal antibody. These observations indicate that calpains regulate gene expression through processing of NF‐κB.
Growth patterns of the humeral and femur length in a multiethnic population
Authors: Raman S. , Teoh T. , Nagaraj S.
Published: July 2002

Objectives: To study whether there are any differences in growth of the femoral and humeral length between the three major ethnic groups in Malaysia viz Malays, Chinese and Indians. The effect on fetal growth by gender of the baby and parity of the mother was also studied. Methods: The setting was the University Hospital, Kuala Lumpur. Thirty‐four Malay, 35 Chinese and 34 Indian normal pregnant middle‐class women were studied longitudinally by monthly ultrasound scans from 18 to 38 weeks of gestation. The data were subjected to regression analysis; the quadratic curve was found to be the most adequate. Dummy variables were used to determine any effects by gender, parity as well as ethnicity on the length of limb growth. There was no difference in birth weights of the three ethnic groups studied, nor in gender or parity. Results: There were found to be significant differences in limb lengths of the Indians (longer) when compared with the Malays and Chinese. Parity seems to affect only Indians in whom the multiparous fetuses have shorter limb lengths than the primiparous. There appears to be no effect by gender. Conclusion: There appear to be definite differences in growth of limb length between the different Malaysian ethnic groups and this should be taken into account when growth charts are used and when fetal weight formulas are calculated using limb lengths. The limitation of this study was that the numbers of subjects studied were small. Larger studies will be able to confirm or refute the findings.
Transvaginal ultrasound, uterine biopsy and hysteroscopy for postmenopausal bleeding
Authors: Giusa‐Chiferi M.G. , Gonçalves W.J. , Baracat E.G. , de Albuquerque Neto L. Cavalcanti , Bortoletto C.C.R. , de Lima G. Rodrigues
Published: July 2002

Objectives: To determine the importance of endometrial biopsy and transvaginal ultrasound in patients with postmenopausal bleeding. Methods: Eighty patients with postmenopausal bleeding were submitted to transvaginal ultrasound followed by endometrial biopsy. Hysteroscopy and dilatation and curettage were carried out to confirm normality of the uterine cavity. Results: The endometrial echo could be visualized in all patients with postmenopausal bleeding. The biopsy failed to detect one case (1.38%) of adenocarcinoma and 14 cases (17.5%) of endometrial polyps. The sensitivity in detecting endometrial malignancy was 94.44% for endometrial biopsy and 100% for transvaginal ultrasound, when the endometrial thickness was more than 8 mm. Conclusions: When the thickness of the endometrial echo is less than 3 mm there is no need for anatomopathologic investigation. When this limit was adopted, all cases were associated with endometrial atrophy, and when the limit was 4 mm or more, active endometria were detected, requiring further histopathologic investigation by hysteroscopy and directed biopsies. Above 8 mm, malignancy may be found.
Expression of basic fibroblast growth factor and its receptors in human fetal microglia cells
Authors: Presta M. , Urbinati C. , Dell'era P. , Lauro G.M. , Sogos V. , Balaci L. , Ennas M.G. , Gremo F.
Published: July 2002

The presence of basic fibroblast growth factor (bFGF) and FGF receptors was investigated in microglia cells derived from human fetal brain long‐term cultures. Production of bFGF was suggested through the capability of microglial extracts to stimulate plasminogen activator (PA) synthesis in endothelial cells. The identity of PA‐stimulating activity with bFGF was confirmed by its high affinity for heparin and its cross‐reactivity with polyclonal antibodies to human recombinant bFGF. These antibodies recognized a cell‐associated Mr 18,000 protein as well as trace amounts of the Mr 24,000 bFGF isoform in Western blot. All microglial cells showed bFGF immunoreactivity in the cytoplasm and, sometimes, in the nucleus.

Scatchard plot analysis of 125I‐bFGF binding data revealed the presence of low affinity heparan‐sulphate proteoglycans (380,000±60,000 sites/cell; Kd = 30±9 pM). Immunocytochemistry confirmed the presence of FGF receptors (10,300+2500 sites/cell; Kd = 30±9 pM). Immunocytochemistry confirmed the presence of FGF receptor (1/flg) on the cell surface of some, but not all microglial cells, with prevalent association to ameboid microglia. Transcripts for FGF receptors 1,2,3 and 4 were found in microglia by Northern blot analysis.

Co‐expression of bFGF and its receptors in human fetal microglia suggests an autocrine role of bFGF in these cells.
Zopiclone's residual effects on psychomotor and information processing skills involved in complex tasks such as car driving: a critical review
Authors: O'Hanlon JF
Published: July 2002

Before and after its introduction in 1987, zopiclone was the object of investigation in 16 psychometric studies employing both healthy volunteers and insomniac patients. Their common purpose was to determine whether nocturnal doses (usually the standard 7.5 mg) possess residual sedative effects that interfere with skilled safety-relevant performance, such as car driving, over the following day. Most studies have found no residual effects. Those that did, have shown them to be modest in magnitude and not to persist for longer than about 12 hours from the time of dosage. Without altering the general conclusion that zopiclone possesses few if any residual effects of clinical relevance, it must be said that the studies reviewed failed to meet current methodological standards and may have left some important questions unanswered.
A novel target of lithium therapy
Authors: Yenush Lynne , Bellés José M. , López-Coronado José M. , Gil-Mascarell Rosario , Serrano Ramón , Rodrı́guez Pedro L.
Published: July 2002

Phosphatases converting 3′‐phosphoadenosine 5′‐phosphate (PAP) into adenosine 5′‐phosphate are of fundamental importance in living cells as the accumulation of PAP is toxic to several cellular systems. These enzymes are lithium‐sensitive and we have characterized a human PAP phosphatase as a potential target of lithium therapy. A cDNA encoding a human enzyme was identified by data base screening, expressed in Escherichia coli and the 33 kDa protein purified to homogeneity. The enzyme exhibits high affinity for PAP (K
m<1 μM) and is sensitive to subtherapeutic concentrations of lithium (IC50=0.3 mM). The human enzyme also hydrolyzes inositol‐1,4‐bisphosphate with high affinity (K
m=0.4 μM), therefore it can be considered as a dual specificity enzyme with high affinity (μM range) for both PAP and inositol‐1,4‐bisphosphate. Hydrolysis of inositol‐1,4‐bisphosphate was also inhibited by lithium (IC50=0.6 mM). Thus, we present experimental evidence for a novel target of lithium therapy, which could explain some of the side effects of this therapy.
Greater diversity than previously thought of chromaffin cell Ca2+ channels, derived from mRNA identification studies
Authors: Garcı́a-Palomero Esther , Cuchillo-Ibáñez Inmaculada , Garcı́a Antonio G. , Renart Jaime , Albillos Almudena , Montiel Carmen
Published: July 2002

Using reverse transcription followed by PCR amplification (RT‐PCR), we have identified multiple messenger RNAs encoding for the neuronal pore‐forming Ca2+ channel subunits α1A (P/Q channel), α1B (N channel), α1D (neuronal/endocrine L channel), α1E (R channel), α1G‐H (T channel) and α1S (skeletal muscle L channel) in bovine chromaffin cells. mRNAs for the auxiliary β2, β3, β4, α2/δ and γ2 subunits were also identified. In agreement with these molecular data, perforated patch‐clamp recordings of whole‐cell Ca2+ currents reveal the existence of functional R‐type Ca2+ channels in these cells that were previously undetected with other techniques. Our results provide a molecular frame for a much wider functional diversity of Ca2+ channels in chromaffin cells than that previously established using pharmacological and electrophysiological approaches.
NADP‐malic enzyme from plants: a ubiquitous enzyme involved in different metabolic pathways
Authors: Drincovich Marı́a F. , Casati Paula , Andreo Carlos S.
Published: July 2002

NADP‐malic enzyme (NADP‐ME) is a widely distributed enzyme that catalyzes the oxidative decarboxylation of L‐malate. Photosynthetic NADP‐MEs are found in C4 bundle sheath chloroplasts and in the cytosol of CAM plants, while non‐photosynthetic NADP‐MEs are either plastidic or cytosolic in various plants. We propose a classification of plant NADP‐MEs based on their physiological function and localization and we describe recent advances in the characterization of each isoform. Based on the alignment of amino acid sequences of plant NADP‐MEs, we identify putative binding sites for the substrates and analyze the phylogenetic origin of each isoform, revealing several features of the molecular evolution of this ubiquitous enzyme.
Constitutive activation of JAK–STAT3 signaling by BRCA1 in human prostate cancer cells
Authors: Gao Bin , Shen Xuening , Kunos George , Meng Qinghui , Goldberg Itzhak D. , Rosen Eliot M. , Fan Saijun
Published: July 2002

Germ‐line mutations of the breast cancer susceptibility gene 1 (BRCA1) confer a high risk for breast and ovarian cancer in women and prostate cancer in men. The BRCA1 protein contributes to cell proliferation, cell cycle regulation, DNA repair and apoptosis; however, the mechanisms underlying these functions of BRCA1 remain largely unknown. Here, we showed that, in Du‐145 human prostate cancer cells, enhanced expression of BRCA1 resulted in constitutive activation of signal transducer and activator transcription factor 3 (STAT3) tyrosine and serine phosphorylation. Moreover, Janus kinase 1 (JAK1) and JAK2, the upstream activators of STAT3, were also activated by BRCA1. Immunoprecipitation assay showed that BRCA1 interacted with JAK1 and JAK2. Blocking STAT3 activation using antisense oligonucleotides significantly inhibited cell proliferation and triggered apoptosis in Du‐145 cells with enhanced expression of BRCA1. These findings indicate that BRCA1 interacts with the components of the JAK–STAT signaling cascade and modulates its activation, which may provide a new critical survival signal for the growth of breast, ovarian and prostate cancers in the presence of normal BRCA1.
Pathways governing G1/S transition and their response to DNA damage
Authors: Bartek Jiri , Lukas Jiri
Published: July 2002

The ability to self‐replicate is a fundamental feature of life, reflected at the cellular level by a highly regulated process initiated in G1 phase via commitment to a round of DNA replication and cell division. Here we briefly highlight recent advances in understanding the molecular pathways which govern the decision of mammalian somatic cells to enter S phase, and the so‐called cell cycle checkpoints which guard the G1/S transition and S phase progression against potentially deleterious effects of genotoxic stress. Particular emphasis is put on the emerging parallel yet cooperative pathways of retinoblastoma protein (pRB)–E2F and Myc, their convergence to control the activity of the cyclin‐dependent kinase 2 (Cdk2) at the G1/S boundary, as well as the two waves of checkpoint responses at G1/S: the rapid pathway(s) leading to Cdc25A degradation, and the delayed p53–p21 cascade, both silencing the Cdk2 activity upon DNA damage.
Expression and characterization of a magnetosome‐associated protein, TPR‐containing MAM22, in Escherichia coli
Authors: Okuda Yoshihiro , Fukumori Yoshihiro
Published: July 2002

A magnetosome‐associated protein, MAM22, contains a TPR domain (five TPR motifs and one putative TPR motif) that has been known to mediate protein–protein interactions. We expressed the mam22 gene in Escherichia coli and found that the purified MAM22 was reversibly self‐aggregated by NaCl. The structural model of MAM22 which has been proposed on the basis of the crystal structure of the N‐terminal TPR domain of a human Ser/Thr protein phosphatase suggests the novel hydrophobic colloidal features of MAM22 with TPR motifs.
Identification and kinetic analysis of the interaction between Nck‐2 and DOCK180
Authors: Tu Yizeng , Kucik Dennis F , Wu Chuanyue
Published: July 2002

Nck‐2 is a newly identified adapter protein comprising three N‐terminal SH3 domains and one C‐terminal SH2 domain. We have identified in a yeast two‐hybrid screen DOCK180, a signaling protein implicated in the regulation of membrane ruffling and migration, as a binding protein for Nck‐2. Surface plasmon resonance analyses reveal that the second and the third SH3 domains interact with the C‐terminal region of DOCK180. The interactions mediated by the individual SH3 domains, however, are much weaker than that of the full length Nck‐2. Furthermore, a point mutation that inactivates the second or the third SH3 domain dramatically reduced the interaction of Nck‐2 with DOCK180, suggesting that both SH3 domains contribute to the DOCK180 binding. A major Nck‐2 binding site, which is recognized primarily by the third SH3 domain, has been mapped to residues 1819–1836 of DOCK180. Two additional, albeit much weaker, Nck‐2 SH3 binding sites are located to DOCK180 residues 1793–1810 and 1835–1852 respectively. Consistent with the mutational studies, kinetic analyses by surface plasmon resonance suggest that two binding events with equilibrium dissociation constants of 4.15±1.9×10−7 M and 3.24±1.9×10−9 M mediate the binding of GST‐Nck‐2 to GST fusion protein containing the C‐terminal region of DOCK180. These studies identify a novel interaction between Nck‐2 and DOCK180. Furthermore, they provide a detailed analysis of a protein complex formation mediated by multiple SH3 domains revealing that tandem SH3 domains significantly enhance the weak interactions mediated by each individual SH3 domain.
Mutations in the fatty acid elongation 1 gene are associated with a loss of β‐ketoacyl‐CoA synthase activity in low erucic acid rapeseed
Authors: Roscoe T.J , Lessire R , Puyaubert J , Renard M , Delseny M
Published: July 2002

Low erucic acid rapeseed (LEAR) is characterised by a near absence of very long chain fatty acids in the seed oil which has been correlated with a lack of acyl‐CoA elongation activity. Here we show that the absence of acyl‐CoA and ATP‐dependent elongation activities in microsomes isolated from LEAR embryos is associated with an absence of β‐ketoacyl‐CoA synthase activity encoded by the Bn‐fatty acid elongation 1 (FAE1) genes. Size exclusion chromatography of solubilised microsomes revealed the presence of a high molecular mass acyl‐CoA elongase complex in high erucic acid rapeseed which was absent in microsomes isolated from LEAR seeds. Although transcripts for the Bn‐FAE1 genes were detected in LEAR embryos, immunoblots using antisera raised against the β‐ketoacyl‐CoA synthase indicated an absence of this protein. Comparison of the deduced amino acid sequences of immature embryo cDNAs reveals that LEAR alleles of Bn‐FAE1 encode variant β‐ketoacyl‐CoA synthase proteins.
The ‘strict’ anaerobe Desulfovibrio gigas contains a membrane‐bound oxygen‐reducing respiratory chain
Authors: Lemos Rita S , Gomes Cláudio M , Santana Margarida , LeGall Jean , Xavier António V , Teixeira Miguel
Published: July 2002

Sulfate‐reducing bacteria are considered as strict anaerobic microorganisms, in spite of the fact that some strains have been shown to tolerate the transient presence of dioxygen. This report shows that membranes from Desulfovibrio gigas grown in fumarate/sulfate contain a respiratory chain fully competent to reduce dioxygen to water. In particular, a membrane‐bound terminal oxygen reductase, of the cytochrome bd family, was isolated, characterized, and shown to completely reduce oxygen to water. This oxidase has two subunits with apparent molecular masses of 40 and 29 kDa. Using NADH or succinate as electron donors, the oxygen respiratory rates of D. gigas membranes are comparable to those of aerobic organisms (3.2 and 29 nmol O2 min−1 mg protein−1, respectively). This ‘strict anaerobic’ bacterium contains all the necessary enzymatic complexes to live aerobically, showing that the relationships between oxygen and anaerobes are much more complex than originally thought.
Shy1p occurs in a high molecular weight complex and is required for efficient assembly of cytochrome c oxidase in yeast
Authors: Nijtmans L.G.J. , Artal Sanz M. , Bucko M. , Farhoud M.H. , Feenstra M. , Hakkaart G.A.J. , Zeviani M. , Grivell L.A.
Published: July 2002

Surf1p is a protein involved in the assembly of mitochondrial respiratory chain complexes. However its exact role in this process remains to be elucidated. We studied SHY1, the yeast homologue of SURF1, with an aim to obtain a better understanding of the molecular pathogenesis of cytochrome c oxidase (COX) deficiency in SURF1 mutant cells from Leigh syndrome patients. Assembly of COX was analysed in a shy1 null mutant strain by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE). Steady‐state levels of the enzyme were found to be strongly reduced, the total amount of assembled complex being approximately 30% of control. The presence of a significant amount of holo‐COX in the SHY1‐disruptant strain suggests that Shy1p may either facilitate assembly of the enzyme, or increase its stability. However, our observations, based on 2D‐PAGE analysis of mitochondria labelled in vitro, now provide the first direct evidence that COX assembly is impaired in a Δshy1 strain. COX enzyme assembled in the absence of Shy1p appears to be structurally and enzymically normal. The in vitro labelling studies additionally indicate that mitochondrial translation is significantly increased in the shy1 null mutant strain, possibly reflecting a compensatory mechanism for reduced respiratory capacity. Protein interactions of both Shy1p and Surf1p are implied by their appearance in a high molecular weight complex of about 250 kDa, as shown by 2D‐PAGE.
Neopterin and 7,8‐dihydroneopterin induce apoptosis in the rat alveolar epithelial cell line L2
Authors: Schobersberger Wolfgang , Hoffmann Georg , Hobisch-Hagen Petra , Böck Günther , Völkl Harald , Baier-Bitterlich Gabriele , Wirleitner Barbara , Wachter Helmut , Fuchs Dietmar
Published: July 2002

The neopterin derivatives, neopterin and 7,8‐dihydroneopterin, modulate the cellular oxidant‐antioxidant balance as well as the expression of the inducible nitric oxide synthase (iNOS) gene. Since apoptosis can be induced by reactive oxygen intermediates and nitric oxide (NO) we investigated whether these neopterin derivatives induce apoptotic cell death. As model we selected the rat alveolar epithelial cell line L2. 24 h incubation of neopterin (1–1000 μM) as well as 7,8‐dihydroneopterin (1–1000 μM) resulted in a significant increase of percent apoptotic cells (measured by FACS analysis). Coincubation of both pteridines with the cytomix (interferon‐γ plus tumor necrosis factor‐α) led to a significantly higher apoptosis than the cytomix alone. In contrast to the cytomix, no iNOS gene expression and no NO release could be detected after incubation with neopterin or 7,8‐dihydroneopterin. We conclude that neopterin and 7,8‐dihydroneopterin are per se inducers of apoptosis which is not mediated by nitric oxide. This may be of importance in inflammatory pulmonary diseases associated with an activation of the cellular immune system.
In vitro low propensity to form nucleosomes of four telomeric sequences
Authors: Cacchione Stefano , Cerone Maria Antonietta , Savino Maria
Published: July 2002

The structural aspects of nucleosome assembly on telomeres are largely unknown. We analyzed by competitive reconstitution the affinities for the histone octamer of telomeric sequences from four different eukaryotic groups, Arabidopsis thaliana, mammals, Tetrahymena, and Saccharomyces cerevisiae. All telomeres reconstitute in nucleosomes with lower association constants than average nucleosomal DNA. DNase I digestion analysis suggests a multiple translational positioning and the lack of rotational positioning, probably due to telomeric repeats length (in most cases 6–8 bp), out of phase with the DNA helical repeat on the nucleosome (10.2 bp). These results could partly explain the lack of nucleosomes on lower eukaryote telomeres, and suggest a high in vivo mobility of telomeric nucleosomes.
Biochemical characterization of cardiotin, a sarcoplasmic reticulum associated protein
Authors: Schaart Gert , Moens Luc , Endert Jorike M , Ramaekers Frans C.S
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Crystallization and preliminary diffraction analysis of a truncated homodimer of human phenylalanine hydroxylase
Authors: Erlandsen Heidi , Martinez Aurora , Knappskog Per M , Haavik Jan , Hough Edward , Flatmark Torgeir
Published: July 2002

A recombinant truncated form (Δ1‐102/Δ428‐452) of the non‐heme iron‐dependent metalloenzyme human phenylalanine hydroxylase (hPAH, phenylalanine 4‐monooxygenase; EC 1.14.16.1) was expressed in E. coli, purified to homogeneity as a homodimer (70 kDa) and crystallized using the hanging drop vapour diffusion method. The crystals are orthorhombic, space group C222 with cell dimensions of a=66.6 Å, b=108.4 Å, c=125.7 Å. The calculated packing parameter (V
m) is 3.24 Å3/Da with four 2‐fold symmetric dimers (or eight momomers) in the unit cell. Data have been collected to 2.0 Å resolution.
Molecular characterization of a novel human PDZ domain protein with homology to INAD from Drosophila melanogaster
Authors: Philipp Stephan , Flockerzi Veit
Published: July 2002

PDZ domains are thought to act as protein‐binding modules mediating the clustering of membrane and membrane‐associated proteins. The INAD protein has been shown to interact via a PDZ domain with the calcium channel TRP which contributes to capacitative calcium entry into Drosophila photoreceptor cells. We have cloned the cDNA of a human INAD‐Like protein (hINADL) of 1524 amino acids in length containing at least five PDZ domains. Additionally, two truncated versions hINADL
Δ304 and hINADL
Δ853 were identified. hInadl transcripts of differing size are expressed in various tissues including brain, where transcripts are abundant in the cerebellum.
Further evidence that the inhibition of glycogen synthase kinase‐3β by IGF‐1 is mediated by PDK1/PKB‐induced phosphorylation of Ser‐9 and not by dephosphorylation of Tyr‐216
Authors: Shaw Morag , Cohen Philip , Alessi Dario R.
Published: July 2002

293 cells were transfected with wild‐type GSK3β (WT‐GSK3β) or a mutant in which the PKB phosphorylation site (Ser‐9) was altered to Ala (A9‐GSK3β). Upon stimulation with IGF‐1 or insulin, WT‐GSK3β was inhibited 75% or 60%, respectively, whereas the activity of the A9‐GSK3β mutant was unaffected. Incubation of WT‐GSK3β with PP2A1 (a Ser/Thr‐specific phosphatase) completely reversed the IGF‐1‐ or insulin‐induced inhibition. IGF‐1 stimulation did not induce any tyrosine dephosphorylation of WT‐GSK3β or A9‐GSK3β. Coexpression of WT‐GSK3β in 293 cells with either PKBα (also known as AKT) or PDK1 (the `upstream' activator of PKB) mimicked the IGF‐1‐ or insulin‐induced phosphorylation of Ser‐9 and inactivation of GSK3β.
Botulinum neurotoxin types A and E require the SNARE motif in SNAP‐25 for proteolysis
Authors: Washbourne Philip , Pellizzari Rossella , Baldini Giulia , Wilson Michael C , Montecucco Cesare
Published: July 2002

Botulinum neurotoxins type A and E (BoNT/A and BoNT/E) are metalloproteases with a unique specificity for SNAP‐25 (synaptosome‐associated protein of 25 kDa), an essential protein component of the neuroexocytotic machinery. It has been suggested that this specificity is directed through the recognition of a nine residue sequence, termed SNARE motif, that is common to the other two SNARE proteins: VAMP (vesicle‐associated membrane protein) and syntaxin, the only known substrates of the other six clostridial neurotoxins. Here we analyse the involvement of the four copies of the SNARE motif present in SNAP‐25 in its interaction with BoNT/A and BoNT/E by following the kinetics of proteolysis of SNAP‐25 mutants deleted of SNARE motifs. We show that a single copy of the motif is sufficient for BoNT/A and BoNT/E to recognise SNAP‐25. While the copy of the motif proximal to the cleavage site is clearly involved in recognition, in its absence, other more distant copies of the motif are able to support proteolysis. Also, a non‐neuronal isoform of SNAP‐25, Syndet, is shown to be sensitive to BoNT/E, but not BoNT/A, whilst the SNAP‐25 isoforms fromTorpedo marmorataandDrosophila melanogasterwere demonstrated not to be substrates of these metalloproteases.
Isolation and characterization of two mutants of human profilin I that do not bind poly(l‐proline)
Authors: Björkegren-Sjögren Camilla , Korenbaum Elena , Nordberg Petra , Lindberg Uno , Karlsson Roger
Published: July 2002

Both mutations caused a reduced affinity for actin, with the W3N replacement having the most pronounced effect. This shows that structural changes in the poly(l‐proline)‐binding region of profilin can affect the distantly located actin‐binding site. Thus, ligands influencing the structure of the poly(l‐proline)‐binding site may regulate actin polymerization through profilin. This is consonant with the finding that PIP2, which changes the tryptophan fluorescence in wild‐type profilin and dissociates the profilin:actin complex in vitro, binds more strongly to the W3N mutant profilin. Thus, the poly(l‐proline)‐binding surface represents a crucial regulatory site of profilin function.
The DNA binding activity of the paired box transcription factor Pax‐3 is rapidly downregulated during neuronal cell differentiation
Authors: Reeves F.C , Fredericks W.J , Rauscher F.J , Lillycrop K.A
Published: July 2002

Mutations in the murine Pax‐3 gene lead to a range of developmental abnormalities including deficiencies in sensory and sympathetic neurones. We have investigated Pax‐3 expression during neuronal differentiation and show levels of Pax‐3 DNA binding decrease upon cell cycle arrest and morphological differentiation. The fall in Pax‐3 DNA binding occurs within 1 h of the induction of differentiation and is mediated in part by a decrease in Pax‐3 mRNA. This decrease in Pax‐3 binding activity precedes any changes in cell proliferation or morphology, suggesting that the downregulation of this transcription factor may be an important prerequisite for the differentiation of neuronal progenitor cells.
Determination of the human c‐Abl consensus DNA binding site
Authors: David-Cordonnier Marie-Hélène , Hamdane Malika , Bailly Christian , D'Halluin Jean-Claude
Published: July 2002

c‐Abl tyrosine kinase, an essential protein of the cell cycle signalling pathways, is implicated in the regulation of RNA polymerase II activity, apoptosis and DNA repair. Its DNA binding activity is important for its biological functions. However, the molecular basis of c‐Abl interaction with DNA remains largely unclear. We delimited the human c‐Abl DNA binding domain and identified its preferred binding site, 5′‐AA/CAACAAA/C. The central AAC motif is highly conserved and constitutes the major core element in the binding sites. EMSAs and footprinting experiments were performed to explore how the c‐Abl fusion protein recognizes specific sequences in DNA.
Immobilized apo‐myoglobin, a new stable reagent for measuring rates of heme dissociation from hemoglobin
Authors: Gattoni Maurizio , Boffi Alberto , Chiancone Emilia
Published: July 2002

Apo‐myoglobin covalently linked on CNBr‐activated Sepharose 4B is proposed as a new heme acceptor for investigating the heme transfer reaction from hemoproteins. Immobilized apo‐myoglobin has the desirable properties of an ideal heme acceptor in that it is characterized by a high affinity for ferric heme, a high stability towards denaturation even at physiological temperatures and can be lyophilized for long‐term storage. The study of heme release from myoglobin at pH 5.0 and 37°C indicates that heme affinity is increased at least 10‐fold relative to the soluble protein. Experiments with human hemoglobin allowed the estimation of the heme release rates from both α and β chains and brought out the greater temperature sensitivity of the α chain heme‐globin linkage.
Prion protein structural features indicate possible relations to signal peptidases
Authors: Glockshuber Rudi , Hornemann Simone , Billeter Martin , Riek Roland , Wider Gerhard , Wüthrich Kurt
Published: July 2002

Transmissible spongiform encephalopathies (TSEs) in mammalian species are believed to be caused by an oligomeric isoform, PrPSc, of the cellular prion protein, PrPC. One of the key questions in TSE research is how the observed accumulation of PrPSc, or possibly the concomitant depletion of PrPC can cause fatal brain damage. Elucidation of the so far unknown function of PrPC is therefore of crucial importance. PrPC is a membrane‐anchored cell surface protein that possesses a so far unique three‐dimensional structure. While the N‐terminal segment 23–120 of PrPC is flexibly disordered, its C‐terminal residues 121–231 form a globular domain with three α‐helices and a two‐stranded β‐sheet. Here we report the observation of structural similarities between the domain of PrP(121–231) and the soluble domains of membrane‐anchored signal peptidases. At the level of the primary structure we find 23% identity and 41% similarity between residues 121–217 of the C‐terminal domain of murine PrP and a catalytic domain of the rat signal peptidase. The invariant PrP residues Tyr‐128 and His‐177 align with the two presumed active‐site residues of signal peptidases and are in close spatial proximity in the three‐dimensional structure of PrP(121–231).
Diadenosine oligophosphates (ApnA), a novel class of signalling molecules?
Authors: Kisselev Lev L , Justesen Just , Wolfson Alexey D , Frolova Lyudmila Yu
Published: July 2002

The diadenosine oligophosphates (Ap
n
A) were discovered in the mid‐sixties in the course of studies on aminoacyl‐tRNA synthetases (aaRS). Now, more than 30 years later, about 300 papers have been published around these substances in attempt to decipher their role in cells. Recently, Ap
n
A have emerged as intracellular and extracellular signalling molecules implicated in the maintenance and regulation of vital cellular functions and become considered as second messengers. Great variety of physiological and pathological effects in mammalian cells was found to be associated with alterations of Ap
n
A levels (n from 2 to 6) and Ap3A/Ap4A ratio. Cell differentiation and apoptosis have substantial and opposite effects on Ap3A/Ap4A ratio in cultured cells. A human Ap3A hydrolase, Fhit, appeared to be involved in protection of cells against tumourigenesis. Ap3A is synthesised by mammalian u synthetase (TrpRS) which in contrast to most other aaRS is unable to synthesise Ap4A and is an interferon‐inducible protein. Moreover, Ap3A appeared to be a preferred substrate for 2‐5A synthetase, also interferon‐inducible, priming the synthesis of 2′ adenylated derivatives of Ap3A, which in turn may serve as substrates of Fhit. Tumour suppressor activity of Fhit is assumed to be associated with involvement of the Fhit·Ap3A complex in cytokine signalling pathway(s) controlling cell proliferation. The Ap
n
A family is potentially a novel class of signal‐transducing molecules whose functions are yet to be determined.
Crystal structure of domain E of Thermus flavus 5S rRNA: a helical RNA structure including a hairpin loop
Authors: Perbandt Markus , Nolte Alexis , Lorenz Siegfried , Bald Rolf , Betzel Christian , Erdmann Volker A
Published: July 2002

The synthetic RNA fragment 5′‐CUGGGCGG(GCGA)CCGCCUGG (nucleotides in parentheses indicate the loop region) corresponds to the natural sequence of domain E from nucleotides 79–97 of the Thermus flavus 5S rRNA including a hairpin loop. The RNA structure determined at 3.0 Å and refined to an R‐value of 24.1% also represents the first X‐ray structure GNRA tetraloop. The loop is in distinctly different conformation from other GNRA tetraloops analyzed by NMR. The conformation of the two molecules in the asymmetric unit is influenced and stabilized by specific intermolecular contacts. The structural features presented here give evidence for the ability of RNA molecules to adapt to specific environments.
Mitochondria and cells produce reactive oxygen species in virtual anaerobiosis: relevance to ceramide‐induced apoptosis
Authors: Degli Esposti Mauro , McLennan Holly
Published: July 2002

Observations of apoptosis in virtual anaerobiosis have raised doubts on the significance of reactive oxygen species in the cascade of events of programmed cell death. This work presents evidence that cells and mitochondrial preparations produce similar levels of hydrogen peroxide under either aerobic or virtually anaerobic conditions. These levels are relevant to the increased production of radicals induced by a ceramide analog that promotes apoptosis. This ceramide acts at center o of mitochondrial complex III.
Genetic analyses of the in vivo function of LolA, a periplasmic chaperone involved in the outer membrane localization of Escherichia coli lipoproteins
Authors: Tajima Terutaka , Yokota Naoko , Matsuyama Shin-ichi , Tokuda Hajime
Published: July 2002

The major outer membrane lipoprotein (Lpp) of Escherichia coli is released from the cytoplasmic membrane into the periplasm as a complex with LolA, a periplasmic chaperone, prior to the localization in the outer membrane. To determine whether or not LolA is generally involved in the outer membrane localization of lipoproteins in vivo, the chromosomal lolA gene was manipulated so as to be controlled by the lac promoter‐operator. Depletion of LolA caused a severe growth defect, and impaired the outer membrane localization of Lpp and Pal, another outer membrane lipoprotein. Although LolA depletion did not immediately arrest the growth of cells lacking Lpp, disruption of the chromosomal lolA gene was lethal to the lpp
− strain, indicating that LolA is generally required for the outer membrane localization of lipoproteins, and therefore essential irrespective of the presence or absence of Lpp.