bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of the environmental implications to include structural changes in a wastewater treatment plant
Authors: Vidal Núria , Poch Manel , Martí Eugènia , Rodríguez‐Roda Ignasi
Published: October 2002

The environmental implications of including structural changes in a wastewater treatment plant to decrease effluent concentrations of nitrogen were evaluated in this study. Environmental effects from these structural changes were assessed by using the Life Cycle Assessment theoretical framework. The wastewater treatment plant selected as a reference scenario had an activated sludge configuration. The Ludzack–Ettinger and Oxidation Ditch configurations were selected as modifications of the reference scenario. Results from this study show that the inclusion of nitrogen removal mechanisms in the configuration of the plant reduces the effect of the plant on the eutrophication, but simultaneously increases the effect on the consumption of abiotic resources, global warming, acidification and human toxicity. These general trends, however, vary depending on the configuration selected to remove nitrogen. Taking all the impacts together, the Oxidation Ditch configuration would cause less environmental impact than the Ludzack–Ettinger configuration, given the characteristics of the selected scenarios.

© 2002 Society of Chemical Industry
Non‐invasive first trimester fetal gender assignment in pregnancies at risk for X‐linked recessive diseases
Authors: Mazza V. , Falcinelli C. , Percesepe A. , Paganelli S. , Volpe A. , Forabosco A.
Published: October 2002

Objectives

Prenatal diagnosis in families affected by X‐linked recessive disorders should ideally be limited to the subjects at increased risk, i.e. male fetuses, in order to avoid the risk of fetal loss due to the invasive procedure in healthy female fetuses. The aim of the study was to assess the fetal sex within the first trimester of gestation by two non‐invasive approaches, using ultrasonography and a molecular analysis of fetal DNA extracted from whole maternal blood with specific markers, in order to avoid invasive sampling in female fetuses.

Methods

A total number of 18 fetuses at risk for an X‐linked recessive disease were included in the present investigation. Maternal peripheral blood was analysed between 7 and 12 weeks of gestation by nested PCR for the detection of fetal DNA and the prediction of fetal gender. In addition, when the biparietal diameter (BPD) was between 21 and 23 mm, an ultrasonographic examination was carried out to assess the fetal gender. CVS was then performed in male fetuses only.

Results

Fetal gender was correctly assigned by ultrasonography between 21 and 23 mm of BPD in all the cases studied, whereas DNA extracted from whole maternal blood accurately predicted the gender in all the female cases (10), but failed in 4 out of 8 male fetuses, erroneously assigned as females.

Conclusion

The present study shows that sonography is able to accurately predict the fetal gender within the first trimester of pregnancy, whereas the molecular analysis of DNA extracted from whole maternal blood is biased by false‐Y‐negative results in 50% of the cases. Copyright © 2002 John Wiley & Sons, Ltd.
Second trimester trisomy 21 maternal serum marker screening. Results of a countrywide study of 854 902 patients
Authors: Muller Françoise , Forestier François , Dingeon Bernard ,   for the ABA Study Group
Published: October 2002

Objectives

In France, maternal serum marker screening is governed by specific legislation. We conducted a study of the countrywide trisomy 21 screening based on second trimester maternal serum markers.

Methods

We reviewed the medical records of 854 902 patients prospectively screened for second trimester maternal serum markers in the 60 authorised laboratories over the two‐year period 1997–1998. All patients screened in France were included. The risk of trisomy 21 was calculated from the combination of maternal age and maternal serum markers. The same cut‐off (1/250) was used in all laboratories.

Results

In 1998, 65% of pregnant women underwent maternal serum screening. In the 837 765 patients under 38 years of age who were screened, 54 321 (6.48%; 5% CI 6.42–6.53%) had a calculated risk >1/250. Of the 884 Down syndrome cases observed, 626 were detected by maternal serum markers (70.8%; 5% CI 67.8–73.8%). These good results can be explained by a strict quality control of all steps. For the 13 891 patients over 38 years of age, the Down syndrome detection rate was 98.9% for a 34% false‐positive rate.

Conclusions

Strict rules covering prenatal trisomy 21 screening are of benefit to patients, practitioners and laboratories alike, and ensure good quality control, a high trisomy 21 detection rate and a low amniocentesis rate. Copyright © 2002 John Wiley & Sons, Ltd.
Can Sporadic Medullary Thyroid Carcinoma Be Biochemically Predicted? Prospective Analysis of 66 Operated Patients with Elevated Serum Calcitonin Levels
Authors: Iacobone Maurizio , Niccoli‐Sire Patricia , Sebag Frederic , De Micco Catherine , Henry Jean‐François
Published: October 2002

Measuring serum calcitonin (CT) in patients with thyroid diseases allows preoperative diagnoses of sporadic medullary thyroid carcinoma (MTC) and C‐cell hyperplasia (CCH). The aim of this prospective study was to distinguish biochemically between CCH and MTC. Basal CT (bCT) was determined in 7276 consecutive patients referred for thyroid disease. Patients with recurrent, persistent, or familial MTC were excluded. When bCT was > 10 pg/ml a pentagastrin‐stimulated CT (sCT) assay was performed. Patients were routinely operated on when bCT > 30 pg/ml or sCT > 100
pg/ml or when other indications for surgery were present. An extensive search for CCH or microscopic MTC was conducted by immunochemistry.
Pathologic findings were correlated with the bCT and sCT values. In this study 66 patients were included. No morphologic alterations of C‐cells were observed in 5 patients; 16 patients presented with CCH and
45 with MTC. Statistical analysis revealed a correlation of sCT and overall bCT with tumor size and staging (p <0.001).
Considering cutoff values for bCT of ≤ 30 pg/ml and for sCT of ≤ 200 pg/ml, the positive predictive value of the test to detect MTC was 100% and the negative predictive value 63%. No patients with MTC at stage 2 to 4 had bCT <30 pg/ml or sCT
<200 pg/ml. A bCT value of ≤ 30 pg/ml or sCT ≤ 200 pg/ml
(or both) is highly predictive of MTC, requiring total thyroidectomy with lymph node dissection. Values of bCT <30 pg/ml and sCT <200 pg/ml do not distinguish between CCH and MTC at stage 1. In this case total thyroidectomy at least is recommended, and the role of nodal dissection might be discussed.
HERC3 binding to and regulation by ubiquitin
Authors: Cruz Cristina , Ventura Francesc , Bartrons Ramon , Rosa Jose Luis
Published: October 2002

Members of the HERC (domain homologous to E6 associated protein carboxy‐terminus and RCC1 domain protein) family may function both as guanine nucleotide exchange factors and E3 ubiquitin ligases. Here we identify an unstudied member, HERC3. This protein was recognized by specific antibodies in different cell types. HERC3 was located in the cytosol and in vesicular‐like structures containing β‐COP, ARF and Rab5 proteins. Involvement of HERC3 in the ubiquitin system was suggested by its ability to interact with ubiquitin. The conserved cysteine in HECT proteins was not essential for this non‐covalent binding. Moreover, HERC3 was a substrate of ubiquitination being degraded by the proteasome. These observations indicate a fine regulation of HERC3 and suggest a role in vesicular traffic and ubiquitin‐dependent processes.
The unusual transhydrogenase of Entamoeba histolytica
Authors: Weston Chris J. , White Scott A. , Jackson J.Baz
Published: October 2002

We have expressed and purified a protein fragment from Entamoeba histolytica. It catalyses transhydrogenation between analogues of NAD(H) and NADP(H). The characteristics of this reaction resemble those of the reaction catalysed by a complex of the NAD(H)‐ and NADP(H)‐binding subunits of proton‐translocating transhydrogenases from bacteria and mammals. It is concluded that the complete En. histolytica protein, which, along with similar proteins from other protozoan parasites, has an unusual subunit organisation, is also a proton‐translocating transhydrogenase. The function of the transhydrogenase, thought to be located in organelles which do not have the enzymes of oxidative phosphorylation, is not clear.
Fatty acid synthesis in developing leaves of Brassica napus in relation to leaf growth and changes in activity of 3‐oxoacyl‐ACP reductase
Authors: O'Hara Paul , Slabas Antoni R , Fawcett Tony
Published: October 2002

In young expanding leaves of Brassica napus, the demand for fatty acids is met by de novo biosynthesis of fatty acid synthase components, as demonstrated by 3‐oxoacyl‐ACP reductase. Using a novel radio‐chemical assay for 3‐oxoacyl‐ACP reductase and specific antibodies, we have demonstrated a direct relationship between the increase in activity and synthesis of polypeptide. The maximum rate of fatty acid synthesis was between 4 and 7 days post‐emergence, but slowed after this point even though 3‐oxoacyl‐ACP reductase activity was high. Leaf area continued to expand in a linear fashion after reductions in both enzyme activity and the rate of fatty acid synthesis.
Rab3a controls exocytosis in cholecystokinin‐secreting cells
Authors: Gevrey Jean-Claude , Laurent Séverine , Saurin Jean-Christophe , Némoz-Gaillard Eric , Regazzi Romano , Chevrier Anne-Marie , Chayvialle Jean-Alain , Abello Jacques
Published: October 2002

The expression of rab3A and rab3D isoforms in the enteroendocrine, cholecystokinin‐secreting, cell lines STC‐1 and GLUTag is here demonstrated. In contrast, rab3B is undetectable in these two cell lines, and rab3C is only slightly expressed in GLUTag cells. Using a transient co‐transfection system with human growth hormone as reporter protein, we show that overexpression of the GTPase‐deficient mutant rab3AQ81L, but not rab3DQ81L, significantly decreases human growth hormone secretory responses to various agonists in STC‐1 cells. These results indicate that endocrine cell lines of intestinal origin express rab3A and rab3D proteins, but the GTP‐bound form of rab3A only acts as a negative modulator in the control of cholecystokinin secretion from STC‐1 cells.
C‐terminal propeptide of the Caldariomyces fumago chloroperoxidase: an intramolecular chaperone?
Authors: Conesa Ana , Weelink Gerri , van den Hondel Cees A.M.J.J. , Punt Peter J.
Published: October 2002

The Caldariomyces fumago chloroperoxidase (CPO) is synthesised as a 372‐aa precursor which undergoes two proteolytic processing events: removal of a 21‐aa N‐terminal signal peptide and of a 52‐aa C‐terminal propeptide. The Aspergillus niger expression system developed for CPO was used to get insight into the function of this C‐terminal propeptide. A. niger transformants expressing a CPO protein from which the C‐terminal propeptide was deleted failed in producing any extracellular CPO activity, although the CPO polypeptide was synthesised. Expression of the full‐length gene in an A. niger strain lacking the KEX2‐like protease PclA also resulted in the production of CPO cross‐reactive material into the culture medium, but no CPO activity. Based on these results, a function of the C‐terminal propeptide in CPO maturation is indicated.
Phospholipid membrane restructuring induced by saposin C: a topographic study using atomic force microscopy
Authors: You Hong Xing , Yu Lei , Qi Xiaoyang
Published: October 2002

The enzymatic activity of glucosylceramidase depends on the presence of saposin C (Sap C) and acidic phospholipid‐containing membranes. In order to delineate the mechanism underlying Sap C stimulation of the enzyme activity, it is important to understand how Sap C interacts with phospholipid membranes. We studied the dynamic process of Sap C interaction with planar phospholipid membranes, in real time, using atomic force microscopy (AFM). The phospholipid membrane underwent restructuring upon addition of Sap C. The topographic characteristics of the membrane restructuring include the appearance of patch‐like new features, initially emerged at the edge of phospholipid membranes and extended laterally with time. Changes in the image contrast of the phospholipid membrane observed after the Sap C addition indicate that a new phase of lipid–protein structure has formed during membrane restructuring. The process of membrane restructuring is dynamic, commencing shortly after Sap C addition, and continuing throughout the duration of AFM imaging (about 30 min, sometimes over 1 h). This study demonstrated the potential of AFM real‐time imaging in studying protein–membrane interactions.
Features of V‐ATPases that distinguish them from F‐ATPases
Authors: Perzov Natalie , Padler-Karavani Vered , Nelson Hannah , Nelson Nathan
Published: October 2002

The general structure of F‐ and V‐ATPases is quite similar and they may share a common mechanism of action that involves mechanochemical energy transduction. Both holoenzymes are composed of catalytic sectors, F1 and V1 respectively, and membrane sectors, Fo and Vo respectively. Although we assume that a similar mechanism underlies ATP‐dependent proton pumping by F‐ and V‐ATPases in eukaryotic cells, the latter cannot catalyze pmf‐driven ATP synthesis. The loss of this ability is probably due to a proton slip that is a consequence of alterations in its membrane sector. The major events include gene duplication of the proteolipids and the presence of three distinct proteolipids in each complex.
Zn2+ binding to the cytoplasmic side of Paracoccus denitrificans cytochrome c oxidase selectively uncouples electron transfer and proton translocation1
Authors: Kannt Aimo , Ostermann Thomas , Müller Hannelore , Ruitenberg Maarten
Published: October 2002

Using a combination of stopped‐flow spectrophotometric proton pumping measurements and time‐resolved potential measurements on black lipid membranes, we have investigated the effect of Zn2+ ions on the proton transfer properties of Paracoccus denitrificans cytochrome c oxidase. When zinc was enclosed in the interior of cytochrome c oxidase containing liposomes, the H/e stoichiometry was found to gradually decrease with increasing Zn2+ concentration. Half‐inhibition of proton pumping was observed at [Zn2+]
i
=75 μM corresponding to about 5–6 Zn2+ ions per oxidase molecule. In addition, there was a significant increase in the respiratory control ratio of the proteoliposomes upon incorporation of Zn2+. Time‐resolved potential measurements on a black lipid membrane showed that the electrogenic phases slowed down in the presence of Zn2+ correspond to phases that have been attributed to proton uptake from the cytoplasmic side and to proton pumping. We conclude that Zn2+ ions bind close to or within the two proton transfer pathways of the bacterial cytochrome c oxidase.
The cysteine residue of the SoxY protein as the active site of protein‐bound sulfur oxidation of Paracoccus pantotrophus GB17
Authors: Quentmeier Armin , Friedrich Cornelius G.
Published: October 2002

Four proteins of Paracoccus pantotrophus are required for hydrogen sulfide‐, sulfur‐, thiosulfate‐ and sulfite‐dependent horse heart cytochrome c reduction. The lack of free intermediates suggested a protein‐bound sulfur oxidation mechanism. The SoxY protein has a novel motif containing a cysteine residue. Electrospray ionization and matrix‐assisted laser desorption ionization mass spectrometry of the SoxYZ protein revealed one mass for SoxZ and different masses for SoxY, indicating native SoxY (10 977 Da) and SoxY with additional masses of +32, +80, +112 and +144 Da, suggesting addition of sulfur, sulfite, thiosulfate and thioperoxomonosulfate. Reduction of SoxY removed the additional masses, indicating a thioether or thioester bond. N‐Ethylmaleimide inhibited thiosulfate‐oxidation and the kinetics suggested a turn‐over‐dependent mode of action. These data were evidence that the sulfur atom to be oxidized was covalently linked to the thiol moiety of the cysteine residue of SoxY and the active site of sulfur oxidation.
Binding specificity of siglec7 to disialogangliosides of renal cell carcinoma: possible role of disialogangliosides in tumor progression
Authors: Ito Akihiro , Handa Kazuko , Withers Donald A. , Satoh Makoto , Hakomori Sen-itiroh
Published: October 2002

Previous studies indicate that expression of higher gangliosides in renal cell carcinoma (RCC) is correlated with metastatic potential, particularly in the lung. Out of five major gangliosides in RCC, three disialogangliosides (disialogalactosylgloboside, IV3NeuAcIII6NeuAcLc4, and IV4GalNAcIV3NeuAcIII6NeuAcLc4) bind strongly to siglec7, which is expressed highly in monocytes and natural killer cells. Out of other gangliosides tested, 2→6 sialylparagloboside, GD3, GD2, and GT1b, but not other lacto‐ or ganglio‐series gangliosides, showed clear binding to siglec7. In view of preferential metastasis of RCC to the lung, and binding of RCC cell line TOS‐1 to lung tissue sections as shown in our previous study, we examined expression of siglec7 in the lung. siglec7 is expressed highly in resident blood cells, but not in parenchymatous cells. TOS‐1 cells aggregate together strongly through adhesion with peripheral blood mononuclear cells to form large clumps. This suggests the possibility that such aggregates may form embolisms of microvasculature, particularly in the lung, which initiate metastasis. Other possible roles of higher gangliosides in RCC in promoting metastasis and tumor progression are discussed.
Molecular dynamics study of aquaporin‐1 water channel in a lipid bilayer
Authors: Zhu Fangqiang , Tajkhorshid Emad , Schulten Klaus
Published: October 2002

The aquaporin‐1 water channel was modeled in a palmitoyl‐oleoyl‐phosphatidyl‐choline lipid bilayer, by means of molecular dynamics simulations. Interaction of the protein with the membrane and inter‐monomer interactions were analyzed. Structural features of the channel important for its biological function, including the Asn‐Pro‐Ala (NPA) motifs, and the diffusion of water molecules into the channels, were investigated. Simulations revealed the formation of single file water inside the channels for certain relative positions of the NPA motifs.
Nitric oxide enhances MPP+ inhibition of complex I
Authors: Cleeter M.W.J. , Cooper J.M. , Schapira A.H.V.
Published: October 2002

There is evidence that 1‐methyl‐4‐phenyl‐1,2,3,6 tetrahydropyridine (MPTP) toxicity is mediated through both inhibition of mitochondrial complex I and free radical generation. 7‐Nitroindazole protects against MPTP toxicity in vitro and in vivo, and this appears to be related to its inhibition of nitric oxide (NO) synthase. We now show that the NO generator, glutathione‐N‐oxide, enhances the inhibitory action of 1‐methyl‐4‐phenylpyridinium (MPP+) on complex I activity in brain submitochondrial particles. We propose that the NO‐induced reversible inhibition of complex IV (cytochrome oxidase) potentiates the MPP+‐induced irreversible free radical‐mediated inhibition of complex I. Thus, NO may ‘prime’ the respiratory chain to the effects of MPP+. These data provide evidence for an interaction between NO and MPP+ at the level of the respiratory chain.
Effect of receptor phosphorylation on the binding between IRS‐1 and IGF‐1R as revealed by surface plasmon resonance biosensor
Authors: Huang Minghui , Lai Wan Ping , Wong Man Sau , Yang Mengsu
Published: October 2002

A receptor binding assay based on the surface plasmon resonance (SPR) biosensor technique was developed to study the interaction between insulin‐like growth factor‐1 receptor (IGF‐1R) and its intracellular substrate protein insulin receptor substrate‐1 (IRS‐1). The sensor surface was modified with anti‐IGF‐1R (α‐subunit) monoclonal antibodies for the capturing of the receptor‐containing membrane fragments from cell lysates. The IGF‐1R was successfully immobilized on the sensor surface with binding capability for its intracellular substrates. SPR measurements showed that the tyrosine phosphorylation of IGF‐1R induced by its extracellular ligand insulin‐like growth factor‐1 caused the receptor to bind with IRS‐1 10 times faster than the unactivated receptor. As a result, the affinity constants of IRS‐1 to phosphorylated and unphosphorylated IGF‐1R were (8.06±5.18)×109 M−1 and (9.81±4.61)×108 M−1, respectively.
Connexin45 directly binds to ZO‐1 and localizes to the tight junction region in epithelial MDCK cells
Authors: Kausalya P.Jaya , Reichert Manuela , Hunziker Walter
Published: October 2002

Zonula occludens protein 1 (ZO‐1) is a cytosolic tight junction protein that tethers transmembrane proteins such as occludin, claudin and junctional adhesion molecule to the actin cytoskeleton. The interaction between ZO‐1 and claudin or junctional adhesion molecule occurs via the amino‐terminal PSD95/Dlg/ZO‐1 (PDZ) domains in ZO‐1. A yeast two‐hybrid screen to search for proteins that interact with the PDZ domains of ZO‐1 identified connexin (Cx) 45. Cx45 interacts with the PDZ domains of ZO‐1 and ZO‐3, but not ZO‐2, via a short C‐terminal PDZ binding motif (SVWI). In transfected epithelial Madin–Darby canine kidney cells, Cx45 co‐localizes with endogenous ZO‐1 at or near tight junctions and co‐precipitation experiments show that Cx45 and ZO‐1 directly interact. Inactivating the C‐terminal PDZ‐binding motif in Cx45 affects its co‐precipitation and co‐localization with ZO‐1. The growing number of connexins (i.e. Cx43 and Cx45) that can associate with ZO proteins indicate that ZO proteins may play a more general role in organizing gap junctions and/or in recruiting signaling molecules that regulate intercellular communication.
Tryptophan‐dependent sensitized photoinactivation of colicin E1 channels in bilayer lipid membranes
Authors: Rokitskaya T.I. , Zakharov S.D. , Antonenko Yu.N. , Kotova E.A. , Cramer W.A.
Published: October 2002

The bacterial toxin colicin E1 is known to induce voltage‐gated currents across a planar bilayer lipid membrane. In the present study, it is shown that the colicin‐induced current decreased substantially upon illumination of the membrane in the presence of the photosensitizer, aluminum phthalocyanine. This effect was almost completely abolished by the singlet oxygen quencher, sodium azide. Using single tryptophan mutants of colicin E1, Trp495 was identified as the amino acid residue responsible for the sensitized photodamage of the colicin channel activity. Thus, the distinct participation of a specific amino acid residue in the sensitized photoinactivation of a defined protein function was demonstrated. It is suggested that Trp495 is critical for the translocation and/or anchoring of the colicin channel domain in the membrane.
The solution structure and heme binding of the presequence of murine 5‐aminolevulinate synthase
Authors: Goodfellow Brian J , Dias Jorge S , Ferreira Glória C , Henklein Peter , Wray Victor , Macedo Anjos L
Published: October 2002

The mitochondrial import of 5‐aminolevulinate synthase (ALAS), the first enzyme of the mammalian heme biosynthetic pathway, requires the N‐terminal presequence. The 49 amino acid presequence transit peptide (psALAS) for murine erythroid ALAS was chemically synthesized, and circular dichroism and 1H nuclear magnetic resonance (NMR) spectroscopies used to determine structural elements in trifluoroethanol/H2O solutions and micellar environments. A well defined amphipathic α‐helix, spanning L22 to F33, was present in psALAS in 50% trifluoroethanol. Further, a short α‐helix, defined by A5–L8, was also apparent in the 26 amino acid N‐terminus peptide, when its structure was determined in sodium dodecyl sulfate. Heme inhibition of ALAS mitochondrial import has been reported to be mediated through cysteine residues in presequence heme regulatory motifs (HRMs). A UV/visible and 1H NMR study of hemin and psALAS indicated that a heme–peptide interaction occurs and demonstrates, for the first time, that heme interacts with the HRMs of psALAS.
A plant Shaker‐like K+ channel switches between two distinct gating modes resulting in either inward‐rectifying or ‘leak’ current
Authors: Dreyer Ingo , Michard Erwan , Lacombe Benoı̂t , Thibaud Jean-Baptiste
Published: October 2002

Among the Shaker‐like plant potassium channels, AKT2 is remarkable because it mediates both instantaneous ‘leak‐like’ and time‐dependent hyperpolarisation‐activated currents. This unique gating behaviour has been analysed in Xenopus oocytes and in COS and Chinese hamster ovary cells. Whole‐cell and single‐channel data show that (i) AKT2 channels display two distinct gating modes, (ii) the gating of a given AKT2 channel can change from one mode to the other and (iii) this conversion is under the control of post‐translational factor(s). This behaviour is strongly reminiscent of that of the KCNK2 channel, recently reported to be controlled by its phosphorylation state.
DNA polymerase β imbalance increases apoptosis and mutagenesis induced by oxidative stress
Authors: Fréchet Mathilde , Canitrot Yvan , Cazaux Christophe , Hoffmann Jean-Sébastien
Published: October 2002

Oxidative stress has been proposed to be one of the major causes leading to the accumulation of mutation that is associated with the initiation and progression of cancers. Elevated expression of DNA polymerase β, an event found in many human tumors, has been shown to generate a mutator phenotype. Here, we demonstrated that overexpression of DNA polymerase β strengthens the mutagenicity of oxidative damages, concomitantly with a higher cellular sensitivity and increased apoptosis. Deregulated expression of DNA polymerase β could represent a predisposition factor for mutagenic effects of oxidative stress and thus have implication in the generation and/or evolution of cancer.
The polymerization mechanism of the bacterial cell division protein FtsZ
Authors: Scheffers Dirk-Jan , Driessen Arnold J.M.
Published: October 2002

Bacteria and archaea usually divide symmetrically by formation of a septum in the middle of the cell. A key event in cell division is the assembly of the FtsZ ring. FtsZ is the prokaryotic homolog of tubulin and forms polymers in the presence of guanine nucleotides. Here, we specifically address the polymerization of FtsZ and the role of nucleotide hydrolysis in polymer formation and stabilization. Recent structural and biochemical results are discussed and a model for FtsZ polymerization, similar to that for tubulin, is presented.
Two distinct isopentenyl diphosphate isomerases in cytosol and plastid are differentially induced by environmental stresses in tobacco
Authors: Nakamura Ayako , Shimada Hiroshi , Masuda Tatsuru , Ohta Hiroyuki , Takamiya Ken-ichiro
Published: October 2002

Two distinct cDNA clones (IPI1 and IPI2) encoding IPI were isolated from Nicotiana tabacum. In situ expression of isopentenyl diphosphate isomerase‐1 (IPI1)– and IPI2–green fluorescent protein fusion constructs revealed that IPI1 and IPI2 were localized in chloroplast and cytosol, respectively. The level of IPI1 mRNA was increased under high‐salt and high‐light stress conditions, while that of IPI2 mRNA was increased under high‐salt and cold stress conditions. Both IPI transcripts were increased in an abscisic acid‐independent manner. This is the first report of a cytosolic IPI. The results indicated that two distinct IPIs were differentially induced in response to stress.
Pim‐1 translocates sorting nexin 6/TRAF4‐associated factor 2 from cytoplasm to nucleus
Authors: Ishibashi Yuki , Maita Hiroshi , Yano Minoru , Koike Naoyuki , Tamai Katsuyuki , Ariga Hiroyoshi , Iguchi-Ariga Sanae M.M.
Published: October 2002

Pim‐1, an oncogene product of serine/threonine kinase, has been found to play roles in apoptosis induction/suppression, cell‐cycle progression and transcriptional regulation by phosphorylating the target proteins involved in these processes. The target proteins phosphorylated by Pim‐1, including p100, Cdc25A, PAP‐1 and heterochromatin protein 1, have been identified. The precise functions of Pim‐1, however, are still poorly understood. In this study, we identified tumor necrosis factor receptor‐associated factor 4‐associated factor 2/sorting nexin 6 (TFAF2/SNX6) as a Pim‐1‐binding protein, and we found that TFAF2/SNX6 was phosphorylated and translocated from the cytoplasm to nucleus by Pim‐1. This translocation of the protein was not affected by Pim‐1‐dependent phosphorylation. Since sorting nexins, including TFAF2/SNX6, have been reported to be located in the cytoplasm or membrane by association with several receptors of tyrosine‐ or serine/threonine‐kinase, this is the first report of TFAF2/SNX6 being located in the nucleus after binding to Pim‐1.
Down‐regulation of N‐myristoyl transferase expression in human T‐cell line CEM by human immunodeficiency virus type‐1 infection
Authors: Takamune Nobutoki , Tanaka Tadahiro , Takeuchi Hiroki , Misumi Shogo , Shoji Shozo
Published: October 2002

The present study focuses on the expression level of N‐myristoyl transferase (NMT) in the course of human immunodeficiency virus type‐1 (HIV‐1) infection. HIV‐1 structural proteins were gradually expressed during the process of infection of the human T‐cell line CEM, whereas the expression levels of NMT subsequently decreased under the same conditions. In addition, the chronically HIV‐1‐infected T‐cell line CEM/LAV‐1 exhibited low expression levels of NMT. We hypothesize that the decrease in the expression level of NMT due to HIV‐1 infection may be related to the virus’ strategy that leads to its persistent replication.
Membrane‐anchored cyclin A2 triggers Cdc2 activation in Xenopus oocyte
Authors: Faivre Jamila , Frank-Vaillant Marie , Poulhe Robert , Mouly Hélène , Bréchot Christian , Sobczak-Thépot Joëlle , Jessus Catherine
Published: October 2002

In Xenopus oocyte, the formation of complexes between neosynthesized cyclins and Cdc2 contributes to Cdc2 kinase activation that triggers meiotic divisions. It has been proposed that cytoplasmic membranes could be involved in this process. To investigate this possibility, we have injected in the oocyte two undegradable human cyclin A2 mutants anchored to the endoplasmic reticulum (ER) membrane. They encode fusion proteins between the truncated cyclin A2‐Δ152 and a viral or cellular ER‐targeting domain. We show that both mutants are fully functional as mitotic cyclins when expressed in Xenopus oocytes, bind Cdc2 and activate M‐phase promoting factor.
TNFα‐induced glutathione depletion lies downstream of cPLA2 in L929 cells
Authors: Hayter Heather L , Pettus Benjamin J , Ito Fumiaki , Obeid Lina M , Hannun Yusuf A
Published: October 2002

Both glutathione (GSH) depletion and arachidonic acid (AA) generation have been shown to regulate sphingomyelin (SM) hydrolysis and are known components in tumor necrosis factor α (TNFα)‐induced cell death. In addition, both have hypothesized direct roles in activation of N‐sphingomyelinase (SMase); however, it is not known whether these are independent pathways of N‐SMase regulation or linked components of a single ordered pathway. This study was aimed at differentiating these possibilities using L929 cells. Depletion of GSH with L‐buthionin‐(S,R)‐sulfoximine (BSO) induced 50% hydrolysis of SM at 12 h. In addition, TNF induced a depletion of GSH, and exogenous addition of GSH blocked TNF‐induced SM hydrolysis as well as TNF‐induced cell death. Together, these results establish GSH upstream of SM hydrolysis and ceramide generation in L929 cells. We next analyzed the L929 variant, C12, which lacks both cytosolic phospholipase A2 (cPLA2) mRNA and protein, in order to determine the relationship of cPLA2 and GSH. TNF did not induce a significant drop in GSH levels in the C12 line. On the other hand, AA alone was capable of inducing a 60% depletion of GSH in C12 cells, suggesting that these cells remain responsive to AA distal to the site of cPLA2. Furthermore, depleting GSH with BSO failed to effect AA release, but caused a drop in SM levels, showing that the defect in these cells was upstream of the GSH drop and SMase activation. When cPLA2 was restored to the C12 line by expression of the cDNA, the resulting CPL4 cells regained sensitivity to TNF. Treatment of the CPL4 cells with TNF resulted in GSH levels dropping to levels near those of the wild‐type L929 cells. These results demonstrate that GSH depletion following TNF treatment in L929 cells is dependent on intact cPLA2 activity, and suggest a pathway in which activation of cPLA2 is required for the oxidation and reduction of GSH levels followed by activation of SMases.
Molecular characterization of a new adult male putative calycin specific to tergal aphrodisiac secretion in the cockroach Leucophaea maderae
Authors: Cornette Richard , Farine Jean-Pierre , Quennedey Brigitte , Brossut Rémy
Published: October 2002

Lma‐p18 is an epicuticular surface protein specific to the tergal gland aphrodisiac secretion of Leucophaea maderae adult males. Native Lma‐p18 was purified and the complete cDNA sequence was determined by RT‐PCR using primers based on Edman degradation fragments. Northern blot and in situ hybridization analyses showed that Lma‐p18 is expressed exclusively in the anterior part of male tergal gland, which is exposed only during sexual behavior. Sequence analysis indicated that Lma‐p18 belongs to the calycin superfamily and is very similar to Lma‐p22, the first known male‐specific tergal protein in L. maderae. Lma‐p18 and Lma‐p22 were proposed to bind different sexually attractive compounds as other calycins.
Geraniol 10‐hydroxylase1, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis
Authors: Collu Graziella , Unver Nehir , Peltenburg-Looman Anja M.G. , van der Heijden Robert , Verpoorte Robert , Memelink Johan
Published: October 2002

Geraniol 10‐hydroxylase (G10H) is a cytochrome P450 monooxygenase involved in the biosynthesis of iridoid monoterpenoids and several classes of monoterpenoid alkaloids found in a diverse range of plant species. Catharanthus roseus (Madagascar periwinkle) contains monoterpenoid indole alkaloids, several of which are pharmaceutically important. Vinblastine and vincristine, for example, find widespread use as anti‐cancer drugs. G10H is thought to play a key regulatory role in terpenoid indole alkaloid biosynthesis. We purified G10H from C. roseus cells. Using degenerate PCR primers based on amino acid sequence information we cloned the corresponding cDNA. The encoded CYP76B6 protein has G10H activity when expressed in C. roseus and yeast cells. The stress hormone methyljasmonate strongly induced G10h gene expression coordinately with other terpenoid indole alkaloid biosynthesis genes in a C. roseus cell culture.
NMR identification of local structural preferences in HIV‐1 protease tethered heterodimer in 6 M guanidine hydrochloride
Authors: Bhavesh Neel S , Panchal Sanjay C , Mittal Rohit , Hosur Ramakrishna V
Published: October 2002

Understanding protein folding requires complete characterization of all the states of the protein present along the folding pathways. For this purpose nuclear magnetic resonance (NMR) has proved to be a very powerful technique because of the great detail it can unravel regarding the structure and dynamics of protein molecules. We report here NMR identification of local structural preferences in human immunodeficiency virus‐1 protease in the ‘unfolded state’. Analyses of the chemical shifts revealed the presence of local structural preferences many of which are native‐like, and there are also some non‐native structural elements. Three‐bond HN–Hα coupling constants that could be measured for some of the N‐terminal and C‐terminal residues are consistent with the native‐like β‐structure. Unusually shifted 15N and amide proton chemical shifts of residues adjacent to some prolines and tryptophans also indicate the presence of some structural elements. These conclusions are supported by amide proton temperature coefficients and nuclear Overhauser enhancement data. The locations of the residues exhibiting preferred structural propensities on the crystal structure of the protein, give useful insights into the folding mechanism of this protein.
A naturally occurring non‐coding fusion transcript derived from scorpion venom gland: implication for the regulation of scorpion toxin gene expression
Authors: Zhu Shunyi , Li Wenxin , Cao Zhijian
Published: October 2002

Scorpion venom glands synthesize and secrete a great number of low molecular mass toxic peptides for prey and defense. Many cDNAs and genomic genes encoding these toxins have been isolated and sequenced. However, their expression regulation mechanism is not yet known at present. During screening of a cDNA library prepared from venom glands of the scorpion Buthus martensii Karsch, we isolated a natural fusion cDNA composed of the 5′‐untranslated region (UTR) and upstream coding sequence of a long‐chain toxin transcript and the downstream coding sequence and 3′‐UTR of a short‐chain toxin transcript. The junction site is just the overlapping region of 11 nucleotides (GGCAAGGAAAT) between the two wild transcripts, and thus leads to the formation of an early stop codon, which will cause premature translation. Based on the above observations, combined with the genomic data, we proposed a characteristic regulation mechanism of scorpion toxin genes, in which trans‐splicing and nonsense mediated mRNA decay are involved.
Nucleoside diphosphate kinase III is localized to the inter‐membrane space in plant mitochondria
Authors: Sweetlove L.J , Mowday B , Hebestreit H.F , Leaver C.J , Millar A.H
Published: October 2002

Three types of nucleoside diphosphate kinases (NDPKs) are found in plants but the intra‐cellular compartmentation of these proteins is not certain, especially the location of the recently identified type III proteins. Through the fractionation of plant mitochondria from potato and Arabidopsis, display of protein profiles by 2D gel electrophoresis, and identification by mass spectrometry, we present the first direct evidence that type III proteins are localized in the inter‐membrane space of plant mitochondria. The possible metabolic functions of NDPK III are discussed in light of its sub‐cellular localization.
A novel dicationic polyamide ligand binds in the DNA minor groove as a dimer
Authors: Zhang Wentao , Dai Yuqin , Schmitz Uli , Bruice Thomas W.
Published: October 2002

We have investigated DNA binding properties of a dicationic polyamide molecule (GL020924) that has exhibited unique protein displacement and gene regulation activities. Fluorescence, thermal melting and electrospray ionization mass spectrometry experiments showed that the binding stoichiometry of GL020924 is 2:1 to various DNA oligomers with 8–11 contiguous A/T bp. In accordance with those findings, circular dichroism experiments showed GL020924 binds as a partially staggered side‐by‐side dimer spanning 10–12 bp. These observations and molecular modeling studies demonstrate that the 2:1 GL020924–DNA complex may exhibit a novel form of stacking orientation involving at least partially parallel peptide groups.
Protein‐binding partners of the tobacco syntaxin NtSyr1
Authors: Kargul Joanna , Gansel Xavier , Tyrrell Matthew , Sticher Liliane , Blatt Michael R.
Published: October 2002

Syntaxins and other SNARE (oluble SF‐ttachment protein ceptor) complex proteins play a key role in the cellular processes of vesicle trafficking, vesicle fusion and secretion. Intriguingly, the SNARE NtSyr1 (=NtSyp121) from Nicotiana tabacum also appears to have a role in signalling evoked by the plant stress hormone abscisic acid. However, partner proteins contributing to its function(s) remain unknown. We used an affinity chromatography approach to identify proteins from tobacco leaf microsomes that directly interact with the hydrophilic (cytosolic) domains of NtSyr1 and report several interacting proteins with sensitivities to the endopeptidase activity of Clostridium botulinum neurotoxins, including one protein that was recognised by αAtSNAP33 antiserum, raised against the Arabidopsis SNAP25 homologue. Treatment of microsomal membrane fractions indicated a protein near 55 kDa was sensitive to proteolysis by BotN/A and BotN/E, yielding degradation products of approximately 34 and 23 kDa. Expressed and purified AtSNAP33 also bound directly to the cytosolic domain of NtSyr1 and was sensitive to proteolysis by these toxins, suggesting that NtSyr1, a tobacco homologue of AtSNAP33, and coordinate SNAREs are likely to associate as partners for function in vivo.
Anandamide amidohydrolase activity, released in the medium by Tetrahymena pyriformis. Identification and partial characterization
Authors: Karava Vivi , Fasia Lambrini , Siafaka-Kapadai Athanassia
Published: October 2002

Anandamide, an endogenous cannabinoid receptor ligand, was rapidly metabolized by Tetrahymena pyriformis in vivo. Metabolic products were mainly phospholipids as well as neutral lipids, including small amounts of free arachidonic acid. Anandamide amidohydrolase activity was detected in the culture medium by the release of [3H]arachidonic acid from [3H]anandamide, in a time‐ and concentration‐dependent manner. Kinetic experiments demonstrated that the released enzyme had an apparent K
m of 3.7 μM and V
max 278 pmol/min/mg protein. Amidohydrolase activity was maximal at pH 9–10, was abolished by phenylmethylsulfonyl fluoride and was Ca2+‐ and Mg2+‐independent. Thus, T. pyriformis is capable of hydrolyzing anandamide in vivo and releasing amidohydrolase activity.
Direct interaction between a membrane domain subunit and a connector subunit in the H+‐translocating NADH‐quinone oxidoreductase
Authors: Di Bernardo Salvatore , Yagi Takao
Published: October 2002

When Paracoccus denitrificans membranes were treated with a crosslinker, m‐maleimidobenzoyl‐N‐hydroxysuccinimide ester (MBS), a cross‐linked product of M
r ∼31 kDa was found which reacted with antibodies against the hydrophobic subunit Nqo7 and the connector subunit Nqo6. NaI treatment of the Paracoccus membranes before, but not after, the crosslinking step prevented the formation of the 31 kDa band. When Nqo7 and Nqo6 were coexpressed in Escherichia coli, both subunits were located in the membrane fraction. MBS treatment of the E. coli membranes generated the 31 kDa band as in the Paracoccus membranes. These results indicate that Nqo7 interacts with probable N2‐binding Nqo6.
Homocysteine attenuates endothelial haem oxygenase‐1 induction by nitric oxide (NO) and hypoxia
Authors: Sawle Philip , Foresti Roberta , Green Colin J , Motterlini Roberto
Published: October 2002

The disrupted metabolism of homocysteine (Hcy) causes hyperhomocysteinemia, a condition associated with the impairment of nitric oxide (NO) bio‐availability, tissue hypoxia and increased risk of vascular disease. Here, we examined how Hcy modulates the induction of the stress protein haem oxygenase‐1 (HO‐1) evoked by NO releasing agents and hypoxia in vascular endothelial cells. We found that Hcy (0.5 mM) markedly reduced the increase in haem oxygenase activity and HO‐1 protein expression induced by sodium nitroprusside (SNP, 0.5 mM) but did not affect HO‐1 activation mediated by S‐nitroso‐N‐acetyl‐penicillamine. Cells pre‐treated with Hcy followed by addition of fresh medium containing SNP still exhibited an augmented haem oxygenase activity. Interestingly, high levels of Hcy were also able to abolish hypoxia‐mediated HO‐1 expression in a concentration‐dependent manner. These novel findings indicate that hyperhomocysteinemia interferes with crucial signaling pathways required by cells to respond and adapt to stressful conditions.
YFH1‐mediated iron homeostasis is independent of mitochondrial respiration
Authors: Chen Opal S , Kaplan Jerry
Published: October 2002

The human gene frataxin and its yeast homolog YFH1 affect mitochondrial function. Deficits in frataxin result in Friedreich ataxia, while deletion of YFH1 results in respiratory incompetence. We determined that as long as respiratory incompetent yeast express Yfh1p they do not accumulate excessive mitochondrial iron. Deletion of YFH1 in respiratory incompetent yeast results in mitochondrial iron accumulation, while the reintroduction of Yfh1p results in mitochondrial iron export. Further, overexpression of Yfh1p has no effect on oxygen consumption in wild‐type yeast grown in either fermentative or respiratory carbon sources. We conclude that the effect of Yfh1p on mitochondrial iron metabolism is independent of respiratory activity.
Phased A‐tracts bind to the α subunit of RNA polymerase with increased affinity at low temperature
Authors: Katayama Seiichi , Matsushita Osamu , Tamai Eiji , Miyata Shigeru , Okabe Akinobu
Published: October 2002

Previously we showed that the expression of a Clostridium perfringens phospholipase C gene (plc) is activated by promoter upstream phased A‐tracts in a low temperature‐dependent manner. In this paper we characterize the interaction between the α subunit of C. perfringens RNA polymerase and the phased A‐tracts. Hydroxyl radical footprinting and fluorescence polarization assaying revealed that the α subunit binds to the minor grooves of the phased A‐tracts through its C‐terminal domain with increased affinity at low temperature. The result provides a molecular mechanism underlying the activation of the plc promoter by the phased A‐tracts.
Overexpression of cdk4/cyclin D1 induces apoptosis in PC12 cells in the presence of trophic support
Authors: Katayama Kazuhiro , Dobashi Yoh , Kitagawa Masatoshi , Kamekura Soichiro , Kawai Masataka , Kadoya Yuichi , Kameya Toru
Published: October 2002

The induction of apoptosis by cell cycle regulator molecules under conditions optimal for exponential growth was examined in rat pheochromocytoma PC12 cells by overexpression of cyclins and cyclin‐dependent kinases (cdks). By flow cytometry and by immunofluorescence, only cells overexpressing cdk4 or cyclin D1 underwent apoptosis, which was not associated with G1‐arrest. Cdk4 kinase activity was significantly higher in cdk4‐, or cyclin D1‐expressing cells. Furthermore, induction of apoptosis by cdk4 was abrogated by co‐transfection of p16INK4, or dominant negative cdk4. These results suggest that upregulation of cdk4 kinase activity is a primary and critical mediator of apoptosis in PC12 cells under physiological conditions.
The role of 2‐methyl‐6‐phytylbenzoquinone methyltransferase in determining tocopherol composition in Synechocystis sp. PCC6803
Authors: Shintani David K. , Cheng Zigang , DellaPenna Dean
Published: October 2002

A putative 2‐methyl‐6‐phytylbenzoquinone (MPBQ) methyltransferase gene, SLL0418, was identified from the Synechocystis PCC6803 genome based on its homology to previously characterized γ‐tocopherol methyltransferases. Genetic and biochemical evidence confirmed open reading frame (ORF) SLL0418 encodes a MPBQ methyltransferase. An SLL0418 partial knockout mutant accumulated β‐tocopherol with no effect in the overall tocopherol content of the cell. In vitro assays of the SLL0418 gene expressed in Escherichia coli showed the enzyme efficiently catalyzes methylation of ring carbon 3 of MPBQ. In addition, the enzyme also catalyzes the methylation of ring carbon 3 of 2‐methyl‐6‐solanylbenzoquinol in the terminal step of plastoquinone biosynthesis.
A common evolutionary origin of two elementary enzyme folds
Authors: Höcker Birte , Schmidt Steffen , Sterner Reinhard
Published: October 2002

The (βα)8‐barrel is the most frequent and most versatile fold among enzymes [Höcker et al., Curr. Opin. Biotechnol. 12 (2001) 376–381; Wierenga, FEBS Lett. 492 (2001) 193–198]. Structural and functional evidence suggests that (βα)8‐barrels evolved from an ancestral half‐barrel, which consisted of four (βα) units stabilized by dimerization [Lang et al., Science 289 (2000) 1546–550; Höcker et al., Nat. Struct. Biol. 8 (2001) 32–36; Gerlt and Babbitt, Nat. Struct. Biol. 8 (2001) 5–7]. Here, by performing a comprehensive database search, we detect a striking and unexpected structural and amino acid sequence similarity between (βα)4 half‐barrels and members of the (βα)5 flavodoxin‐like fold. These findings provoke the hypothesis that a large fraction of the modern‐day enzymes evolved from a basic structural building block, which can be identified by a combination of sequence and structural analyses.