bluuebunny/crossref_metadata_2025 huggingface dataset:
Proliferation of C6 glioma cells is blunted by the increase in gap junction communication caused by tolbutamide
Authors: Sánchez-Alvarez Rosa , Tabernero Arantxa , Sánchez-Abarca Luis I. , Orfao Alberto , Giaume Christian , Medina José M.
Published: October 2002

We have previously reported that tolbutamide prevents the inhibition of gap junction communication in astrocytes. Here, we show that tolbutamide increases gap junction communication and connexin 43 expression in poorly coupled C6 glioma cells. The increase in communication is concurrent with the inhibition of the rate of proliferation due to a block of the progression of C6 glioma cells through the S phase of the cell cycle. The effects of tolbutamide were quantitatively similar to that found after the elevation of intracellular cAMP. Furthermore, the effects of tolbutamide and cAMP were additive. The possible beneficial effect of tolbutamide on gene therapy for gliomas is discussed.
Exogenous ACC enhances volatiles production mediated by jasmonic acid in lima bean leaves
Authors: Horiuchi Jun-ichiro , Arimura Gen-ichiro , Ozawa Rika , Shimoda Takeshi , Takabayashi Junji , Nishioka Takaaki
Published: October 2002

We report the synergistic effects of exogenous 1‐aminocyclopropane‐1‐carboxylic acid (ACC) and jasmonic acid (JA) on production of induced volatiles by excised lima bean leaves. Application of ACC alone to leaves induced trace amounts of volatiles. ACC positively affected three JA‐induced volatiles, (E)‐ and (Z)‐β‐ocimene, and (Z)‐3‐hexenyl acetate. The ethylene inhibitor, silver thiosulfate, inhibited the production of these compounds. The results suggest synergistic effects of JA and ACC on inducible volatile production by lima bean leaves. Furthermore, lima bean leaves treated with JA plus ACC became more attractive to predatory mites, Phytoseiulus persimilis, than those treated with JA alone.
Dielectric relaxation in a single tryptophan protein
Authors: Ghose Manasi , Mandal Surekha , Roy Debjani , Mandal Radha K. , Basu Gautam
Published: October 2002

Although dielectric relaxation can significantly affect the intrinsic fluorescence properties of a protein, usually it is fast compared to fluorescence timescales and needs to be slowed down by adding viscogens or lowering temperature before its impact on fluorescence can be studied. We report here a remarkable blue shift in fluorescence upon bimolecular quenching in the single‐tryptophan thermostable protein Bj2S, the 2S seed albumin from Brassica juncea, at ambient temperature and viscosity. The magnitude of the blue shift (∼5 nm at 50% quenching by acrylamide) is striking in a single‐tryptophan protein and is attributed to a slowly relaxing dielectric environment in Bj2S from red edge excitation, steady‐state polarization and time‐resolved fluorescence experiments. Our results have important implications on interpretation of fluorescence of proteins with highly constrained backbones and in designing model systems for studying slow protein solvation dynamics using Trp fluorescence as the reporter probe.
A novel progestogen receptor subtype in the Japanese eel, Anguilla japonica1
Authors: Ikeuchi Toshitaka , Todo Takashi , Kobayashi Tohru , Nagahama Yoshitaka
Published: October 2002

A cDNA encoding a second type of a progestogen receptor (ePR2) was isolated from the same library as we had previously cloned a functional PR (ePR1) in eel testis. The amino acid sequence of the ePR2 shows low homology with ePR1 (34%), but both PRs showed progestogen‐dependent transactivation in transfection experiment. Tissue distribution of ePR2 mRNA was clearly different from that of ePR1. Protein interaction between two PRs was demonstrated in vitro by a glutathione S‐transferase pull‐down assay. These results indicate that ePR2 is also a functional PR. This is the first isolation of two different functional PR molecules from a vertebrate.
p62 forms a ternary complex with PKCζ and PAR‐4 and antagonizes PAR‐4‐induced PKCζ inhibition
Authors: Chang Suhwan , Kim Jung Hoe , Shin Jaekyoon
Published: October 2002

It has been reported that prostate apoptosis response‐4 (PAR‐4) binds to and inhibits protein kinase Cζ (PKCζ) which phosphorylates IκB kinase β (IKKβ) for nuclear factor κB (NFκB) activation, while p62 binds to and recruits PKCζ to the NFκB signaling complex. Thus, a mechanism to coordinate the two binding proteins for the regulation of PKCζ is expected to exist. The present data show that p62 and PAR‐4 do not compete for PKCζ binding but directly interact each other and form a ternary complex with PKCζ. Furthermore, p62 not only enhances the catalytic activity of PKCζ but also reactivates catalytically inactive PAR‐4‐bound PKCζ. As the result, over‐expression of p62 protects cells from PAR‐4‐mediated inactivation of NFκB and apoptotic death. Thus, the regulatory role of p62 for free and PAR‐4‐bound PKCζ is important in activation of NFκB.
Genome‐wide detection and family clustering of ion channels
Authors: Harte Rachel , Ouzounis Christos A
Published: October 2002

Ion channels represent an important class of molecules that can be classified into 13 distinct groups. We present a strategy using a ‘learning set’ of well‐annotated ion channel sequences to detect homologues in 32 entire genome sequences from Archaea, Bacteria and Eukarya. A total of 299 putative ion channel protein sequences were detected, with significant variations across species. The clustering of these sequences reveals complex relationships between the different ion channel families.
The observation of excited‐state dynamical evolution in light‐harvesting complex LH2 from Rhodobacter sphaeroides 601
Authors: Guo Lijun , Liu Yuan , Yang Yi , Mi Jun , Xu Can , Xu Chunhe , Qian Shixiong
Published: October 2002

With selective excitation around BChl–B800 and BChl–B850 absorption bands, we observed the evolution of excited‐state dynamics in LH2 from Rhodobacter sphaeroides 601. The dynamical traces demonstrate a dominant excited‐state absorption (ESA) followed concomitantly by an ultrafast transmission increase and decay with pulse‐width limited time scale at 818 nm and 828 nm excitation. The ESA occurring prior to excitonic thermalization or ground‐state bleach was observed at 840 nm as well. These experimental results indicate the competition between the transition from excitonic states to higher‐lying excited states and interexciton relaxation, which are of physical significance for understanding excitation transfer and related mechanisms in LH2.
Lectin‐like oxidized LDL receptor‐1 (LOX‐1) supports adhesion of mononuclear leukocytes and a monocyte‐like cell line THP‐1 cells under static and flow conditions
Authors: Hayashida Kazutaka , Kume Noriaki , Minami Manabu , Kita Toru
Published: October 2002

Adhesion of mononuclear leukocytes to vascular endothelial cells appears one of the initial steps in the process of atherogenesis and inflammation. We examined if LOX‐1, an endothelial scavenger receptor with C‐type lectin‐like structure, can support adhesion of mononuclear leukocytes. Under a static condition, CHO‐K1 cells stably expressing LOX‐1 showed more prominent adhesion of human peripheral blood mononuclear leukocytes and THP‐1 cells than untransfected CHO‐K1 cells, in a temperature‐independent fashion. Mononuclear leukocytes also adhered to plastic plates precoated with recombinant soluble LOX‐1 extracellular domain. A neutralizing anti‐LOX‐1 monoclonal antibody, as well as oxidized low‐density lipoprotein, significantly blocked adhesion of THP‐1 cells to CHO‐K1 cells overexpressing LOX‐1 and bovine aortic endothelial cells. Under a flow condition, increased numbers of THP‐1 cells showed rolling with reduced velocities on LOX‐1‐expressing CHO‐K1 cells, compared with those on untransfected CHO‐K1 cells. Taken together, LOX‐1 can work as a cell surface receptor for mononuclear leukocytes under both static and flow conditions.
Sendai virus C protein impairs both phosphorylation and dephosphorylation processes of Stat1
Authors: Komatsu Takayuki , Takeuchi Kenji , Yokoo Junko , Gotoh Bin
Published: October 2002

Sendai virus expresses C protein that blocks interferon (IFN) signaling. We previously reported suppression of IFN‐stimulated tyrosine phosphorylation of signal transducers and activators of transcription (Stats) in infected cells. However this conclusion has remained controversial. To settle it, we re‐examined the effect of C protein expression on phosphorylation of Stat1 in detail. IFN‐stimulated tyrosine phosphorylation of Stat1 was doubtlessly suppressed early in infection, but the suppression was incomplete, suggesting the importance of the unknown blocking mechanism that inactivates the tyrosine‐phosphorylated (pY)‐Stat1 generated as the signaling leak. Interestingly, the dephosphorylation process of pY‐Stat1 was also impaired. These effects on both phosphorylation and dephosphorylation processes were attributable to the function of the C protein.
The orientation of the antibiotic peptide maculatin 1.1 in DMPG and DMPC lipid bilayers. Support for a pore‐forming mechanism
Authors: Chia C.S.B , Torres J , Cooper M.A , Arkin I.T , Bowie J.H
Published: October 2002

Maculatin 1.1 is an antimicrobial peptide isolated from the Australian tree frog Litoria genimaculata that adopts an amphipathic, α‐helical structure in solution. Its orientation and conformation when incorporated to pre‐formed DMPG (1,2‐dimyristoyl‐sn‐glycero‐3‐phosphoglycerol) and DMPC (1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine) vesicles was determined using polarised Fourier transform infrared–attenuated total reflection infrared and deuterium exchange experiments. For DMPG membranes, our results show insertion of ∼70% of the maculatin 1.1 molecules, with an angle of insertion of approximately 35° to the membrane normal and with a predominant α‐helical structure. These results suggest that maculatin 1.1 acts through a pore‐forming mechanism to lyse bacterial membranes. A similar degree of insertion in DMPG (65%) and α‐helical structure was observed for a biologically inactive, less amphipathic maculatin 1.1 analogue, P15A, although the helix tilt was found to be greater (46°) than for maculatin 1.1. Similar experiments performed using DMPC liposomes showed poor insertion, less than 5%, for both maculatin 1.1 and its analogue. In addition, the shape of the amide I band in these samples is consistent with α‐helix, β‐structure and disordered structures being present in similar proportion. These results clearly show that maculatin 1.1 inserts preferentially in negatively charged membranes (DMPG) which mimic the negatively charged membrane of Gram‐positive bacteria. We attribute the high percentage of insertion of the biologically inactive analogue in DMPG to the fact that its concentration on the membrane surface in our experiments is likely to be much higher than that found in physiological conditions.
The first room‐temperature X‐ray absorption spectra of higher oxidation states of the tetra‐manganese complex of photosystem II
Authors: Haumann Michael , Grabolle Markus , Neisius Thomas , Dau Holger
Published: October 2002

The manganese (Mn) complex of photosystem II catalyzes water oxidation. For the first time, its advancement through the reaction cycle was monitored by time‐resolved X‐ray absorption measurements at the Mn K‐edge at room temperature. The complex was stepped through its four oxidation states by nano‐second‐laser flashes applied to samples exposed to the X‐ray beam. Time courses of the X‐ray fluorescence intensity were recorded during a flash sequence. Extended X‐ray absorption fine‐structure spectra were recorded with the S1, S2, and S3 oxidation states highly populated. The room temperature data is compatible with the formation of a third di‐μ‐oxo bridge between two Mn atoms upon the S2→S3 transition.
Molecular aspects of photodynamic therapy: low energy pre‐sensitization of hypericin‐loaded human endometrial carcinoma cells enhances photo‐tolerance, alters gene expression and affects the cell cycle
Authors: Varriale Linda , Coppola Eva , Quarto Maria , Maria Veneziani Bianca , Palumbo Giuseppe
Published: October 2002

Photosensitization of HEC1‐B cells with a low concentration of hypericin and doses of light below 10 J/cm2 caused cell death (apoptosis occurred mainly at doses between 2 and 5 J/cm2, whereas necrosis prevailed above 6 J/cm2). However, pre‐exposure of cells to innocuous irradiation (2 J/cm2) and successive challenge with a light dose that normally induced apoptosis (5 J/cm2) altered the expression of the proteins involved in the regulation of apoptosis, stress response and cell cycle. This change resulted in a significant increase in cell photo‐tolerance.
Evidence for diversity of substrate specificity among members of the chymase family of serine proteases
Authors: Solivan Suzanne , Selwood Trevor , Wang Zhe-Mei , Schechter Norman M
Published: October 2002

The term chymase is used to signify a chymotrypsin‐like protease stored within the secretory granules of mast cells. Primarily based on amino acid sequence homology, 18 chymases have been identified among different animals. This study, which compares the structure of the primary specificity pocket (S1 subsite), defines a subgroup of four chymases likely to have a substrate specificity with more elastase‐ than chymotrypsin‐like qualities. This difference is due, primarily, to finding a Val instead of a Gly at residue 199, a position corresponding to Gly216 in bovine chymotrypsin and Val216 in neutrophil and porcine elastases. Chymases with Val at 199 are found only in animals expressing multiple chymases, consistent with the premise that their substrate specificity differs from that of chymases with Gly at 199.
Evidence for a functional nitric oxide synthase system in brown adipocyte nucleus
Authors: Giordano Antonio , Tonello Cristina , Bulbarelli Alessandra , Cozzi Valeria , Cinti Saverio , Carruba Michele O , Nisoli Enzo
Published: October 2002

The intracellular localization and activity of the nitric oxide synthase (NOS) isoforms were investigated in rat brown adipocytes. Immunohistochemistry showed cytoplasmic and nuclear staining for the endothelial NOS (eNOS) and inducible NOS (iNOS) isoforms; accordingly, anti‐L‐citrulline antibody, a marker of NOS activity, immunostained both the cytoplasm and the nucleus. The presence of metabolically active NOS in the nucleus was further confirmed by immunoblotting analyses of subcellular fractions of homogenates from cultured brown adipocytes and by measurements of NOS activity in the cytosol and nucleus. Sympathetic stimulation in vivo (i.e. cold exposure or β3‐adrenergic agonist treatment) and in vitro (i.e. noradrenaline treatment of cultured cells) significantly increased both cytosolic and nuclear eNOS and iNOS expression and activities. By contrast, the number of iNOS‐positive, but not eNOS‐positive, nuclei was significantly lower in the functionally impaired brown fat of genetically obese Zucker fa/fa rats. These data suggest the existence of a noradrenaline‐modulated functional NOS system in the nucleus of brown adipocytes.
First patch, then catch: measuring the activity and the mRNA transcripts of a proton pump in individual Lilium pollen protoplasts1
Authors: Gehwolf Renate , Griessner Martin , Pertl Heidi , Obermeyer Gerhard
Published: October 2002

Combining the patch‐clamp method with single‐cell reverse transcription polymerase chain reaction (scRT‐PCR) a fusicoccin‐induced current reflecting the activity of the plasma membrane H+ ATPase of lily pollen protoplasts was measured and subsequently, the ATPase‐encoding mRNAs were collected and amplified. Southern blot signals were observed in all ‘patch–catch’ experiments and could be detected even in 2560‐fold dilutions of the pollen contents. H+ ATPase mRNAs were detectable only in the vegetative but not in the generative cell of pollen as confirmed by immunolocalisation. In 15% of the scRT‐PCR experiments, a random non‐reproducibility of the PCR was observed, probably caused by varying amounts of ATPase mRNAs in the protoplasts.
Molecular cloning and characterization of Kv6.3, a novel modulatory subunit for voltage‐gated K+ channel Kv2.11
Authors: Sano Yorikata , Mochizuki Shinobu , Miyake Akira , Kitada Chika , Inamura Kohei , Yokoi Hiromichi , Nozawa Katsura , Matsushime Hitoshi , Furuichi Kiyoshi
Published: October 2002

We report identification and characterization of Kv6.3, a novel member of the voltage‐gated K+ channel. Reverse transcriptase‐polymerase chain reaction analysis indicated that Kv6.3 was highly expressed in the brain. Electrophysiological studies indicated that homomultimeric Kv6.3 did not yield a functional voltage‐gated ion channel. When Kv6.3 and Kv2.1 were co‐expressed, the heteromultimeric channels displayed the decreased rate of deactivation compared to the homomultimeric Kv2.1 channels. Immunoprecipitation studies indicated that Kv6.3 bound with Kv2.1 in co‐transfected cells. These results indicate that Kv6.3 is a novel member of the voltage‐gated K+ channel which functions as a modulatory subunit.
The glycan core of GPI‐anchored proteins modulates aerolysin binding but is not sufficient: the polypeptide moiety is required for the toxin–receptor interaction
Authors: Abrami Laurence , Velluz Marie-Claire , Hong Yeongjin , Ohishi Kazuhito , Mehlert Angela , Ferguson Michael , Kinoshita Taroh , Gisou van der Goot F
Published: October 2002

Sensitivity of mammalian cells to the bacterial toxin aerolysin is due to the presence at their surface of glycosylphosphatidyl inositol (GPI)‐anchored proteins which act as receptors. Using a panel of mutants that are affected in the GPI biosynthetic pathway and Trypanosoma brucei variant surface glycoproteins, we show that addition of an ethanolamine phosphate residue on the first mannose of the glycan core does not affect binding. In contrast, the addition of a side chain of up to four galactose residues at position 3 of this same mannose leads to an increase in binding. However, protein free GPIs, which accumulate in mutant cells deficient in the transamidase that transfers the protein to the pre‐formed GPI‐anchor, were unable to bind the toxin indicating a requirement for the polypeptide moiety, the nature and size of which seem of little importance although two exceptions have been identified.
Regulation of fibronectin alternative splicing by a basement membrane‐like extracellular matrix
Authors: Srebrow Anabella , Blaustein Matı́as , Kornblihtt Alberto R
Published: October 2002

Hepatocytes are the source of plasma fibronectin (FN) which lacks the alternatively spliced EDI segment, distinctive of oncofetal FN. When hepatic or other epithelial cells are cultured on plastic, EDI inclusion is triggered. Here we report that EDI inclusion is inhibited when hepatic cells are cultured on a basement membrane‐like extracellular matrix (ECM), demonstrating a new role for the ECM in the control of gene expression. The effect is duplicated by collagen IV and laminin but not by collagen I; is not observed with another alternatively spliced FN exon (EDII); and correlates with a decrease in cell proliferation, consistently with high EDI inclusion levels observed in many physiological and pathological proliferative processes.
N6‐Adenine DNA‐methyltransferase in wheat seedlings
Authors: Fedoreyeva Larisa I. , Vanyushin Boris F.
Published: October 2002

The N
6‐adenine DNA‐methyltransferase was isolated from the vacuolar vesicle fraction of wheat coleoptiles. In the presence of S‐adenosyl‐L‐methionine the enzyme de novo methylates the first adenine residue in the TGATCA sequence in the single‐ or double‐stranded DNA substrates but it prefers single‐stranded structures. Wheat adenine DNA‐methyltransferase (wadmtase) is a Mg2+‐ or Ca2+‐dependent enzyme with a maximum activity at pH 7.5–8.0. Wadmtase seems to be responsible for mitochondrial DNA modification that might be involved in the regulation of replication of mitochondria in plants.
Leptin directly stimulates thermogenesis in skeletal muscle
Authors: Dulloo Abdul G , Stock Michael J , Solinas Giovanni , Boss Olivier , Montani Jean-Pierre , Seydoux Josiane
Published: October 2002

Using a method involving repeated oxygen uptake (MO2) determinations in skeletal muscle ex vivo, the addition of leptin was found to increase MO2 in soleus muscles from lean mice. These effects were found to be inhibited by phosphatidylinositol 3‐kinase inhibitors, absent in muscles from obese Leprdb mice which have the dysfunctional long form of leptin receptor, and blunted in muscles from diet‐induced obese mice in the fed state but not during fasting. These findings indicate that leptin has direct thermogenic effects in skeletal muscle, and that these effects require both the long form of leptin receptors and phosphatidylinositol 3‐kinase signalling.
Dephosphorylation of PKCδ by protein phosphatase 2Ac and its inhibition by nucleotides
Authors: Srivastava Jyoti , Goris Jozef , Dilworth Stephen M , Parker Peter J
Published: October 2002

The protein phosphatases PP1c, PP2Ac and PP2Cα are shown to dephosphorylate protein kinase Cδ (PKCδ) in vitro; of these PP2Ac displayed the highest specific activity towards PKCδ. The role of PP2Ac in the dephosphorylation of PKCδ in cells was supported by the demonstration that these proteins could be co‐immunoprecipitated from NIH3T3 cells. However the observation that binding of Mg‐ATP to PKCδ could protect the enzyme from dephosphorylation by PP2Ac in vitro indicates that an additional input/factor is required for dephosphorylation in vivo.
Modelling Alzheimer‐specific abnormal Tau phosphorylation independently of GSK3β and PKA kinase activities
Authors: Delobel P , Flament S , Hamdane M , Delacourte A , Vilain J.P , Buée L
Published: October 2002

In Alzheimer's disease, neurofibrillary degeneration results from the aggregation of abnormally phosphorylated Tau proteins into paired helical filaments. These Tau variants displayed specific epitopes that are immunoreactive with anti‐phospho‐Tau antibodies such as AT100. As shown in in vitro experiments, glycogen synthase kinase 3 β (GSK3β) and protein kinase A (PKA) may be key kinases in these phosphorylation events. In the present study, Tau was microinjected into Xenopus oocytes. Surprisingly, in this system, AT100 was generated without any GSK3β and PKA contribution during the progesterone or insulin‐induced maturation process. Our results demonstrate that a non‐modified physiological process in a cell model can generate the most specific Alzheimer epitope of Tau pathology.
A specific role for tocopherol and of chemical singlet oxygen quenchers in the maintenance of photosystem II structure and function inChlamydomonas reinhardtii
Authors: Trebst Achim , Depka Brigitte , Holländer-Czytko Heike
Published: October 2002

α‐Tocopherol concentrations were determined at low and high light intensities and compared with the rate of photosynthesis, photosystem II (PS II) and its reaction center D1 protein. Blocking of tocopherol biosynthesis at the 4‐hydroxyphenylpyruvate dioxygenase by the herbicide pyrazolynate led to a quick disappearance of α‐tocopherol in high light, as well as of PS II activity and the D1 protein. Homogentisic acid rescued all activities. It is concluded that α‐tocopherol has a continuous turnover as a scavenger of the singlet oxygen that arises from the quenching by oxygen of the triplet of the PS II reaction center and triggers the degradation of the D1 protein. Thus tocopherols are essential to keep photosynthesis active. We suggest that this is why plants make and need tocopherols. Chemical quenchers of singlet oxygen, notably diphenylamines, completely protect PS II, prevent D1 protein degradation and keep tocopherol levels even at very high light intensities. This supports the notion that1O2is the intermediate in light triggered D1 protein turnover.
Conventional protein kinase C isoforms regulate human dopamine transporter activity in Xenopus oocytes
Authors: Doolen Suzanne , Zahniser Nancy R
Published: October 2002

The hypothesis that specific protein kinase C (PKC) isoforms regulate dopamine transporter (DAT) function was tested in Xenopus laevis oocytes expressing human (h)DAT. Activation of conventional PKCs (cPKCs) and novel PKCs (nPKCs) using 10 nM phorbol 12‐myristate 13‐acetate (PMA) significantly inhibited DAT‐associated transport currents. This effect was reversed by isoform‐non‐selective PKC inhibitors, selective inhibitors of cPKCs and δPKC, and by Ca2+ chelation. By contrast, the ϵPKC translocation inhibitor peptide had no effect on PMA‐induced inhibition of hDAT transport‐associated currents. Thus, the primary mechanism by which PMA regulates hDAT expressed in oocytes appears to be by activating cPKC(s).
The roles of hydrogenases 3 and 4, and the F0F1‐ATPase, in H2 production by Escherichia coli at alkaline and acidic pH
Authors: Bagramyan Karine , Mnatsakanyan Nelli , Poladian Anna , Vassilian Anait , Trchounian Armen
Published: October 2002

The hyc operon of Escherichia coli encodes the H2‐evolving hydrogenase 3 (Hyd‐3) complex that, in conjunction with formate dehydrogenase H (Fdh‐H), constitutes a membrane‐associated formate hydrogenlyase (FHL) catalyzing the disproportionation of formate to CO2 and H2 during fermentative growth at low pH. Recently, an operon (hyf) encoding a potential second H2‐evolving hydrogenase (Hyd‐4) was identified in E. coli. In this study the roles of the hyc‐ and hyf‐encoded systems in formate‐dependent H2 production and Fdh‐H activity have been investigated. In cells grown on glucose under fermentative conditions at slightly acidic pH the production of H2 was mostly Hyd‐3‐ and Fdh‐H‐dependent, and Fdh‐H activity was also mainly Hyd‐3‐dependent. However, at slightly alkaline pH, H2 production was found to be largely Hyd‐4, Fdh‐H and F0F1‐ATPase‐dependent, and Fdh‐H activity was partially dependent on Hyd‐4 and F0F1‐ATPase. These results suggest that, at slightly alkaline pH, H2 production and Fdh‐H activity are dependent on both the F0F1‐ATPase and a novel FHL, designated FHL‐2, which is composed of Hyd‐4 and Fdh‐H, and is driven by a proton gradient established by the F0F1‐ATPase.
CLK‐1 protein has DNA binding activity specific to OL region of mitochondrial DNA
Authors: Gorbunova Vera , Seluanov Andrei
Published: October 2002

Mutations in the clk‐1 gene of Caenorhabditis elegans extend worm life span and slow down a variety of physiological processes. Here we report that C. elegans CLK‐1 as well as its mouse homologue have DNA binding activity that is specific to the OL region of mitochondrial DNA. DNA binding activity of CLK‐1 is inhibited by ADP, and is altered by mutations that extend nematode life span. Our results suggest that, in addition to its enzymatic function in ubiquinone biosynthesis, CLK‐1 is involved in the regulation of mtDNA replication or transcription.
Amyloid fibrils from the mammalian protein prothymosin α
Authors: Pavlov Nikolai A. , Cherny Dmitry I. , Heim Gudrun , Jovin Thomas M. , Subramaniam Vinod
Published: October 2002

Mammalian prothymosin α, a small (12 kDa) and extremely acidic protein (pI 3.5), is a member of the growing family of ‘natively’ unfolded proteins. We demonstrate that at low pH (∼3) and high concentrations, prothymosin α is capable of forming regular elongated fibrils with flat ribbon structure 4–5 nm in height and 12–13 nm in width as judged from scanning force and electron microscopy. These aggregates induced a characteristic spectral shift of thioflavin T fluorescence and their circular dichroism spectra were indicative of significant β‐sheet content, suggesting formation of classical amyloid. Our findings indicate that natively unfolded proteins may have a general propensity to form amyloid fibrils under conditions inducing partially folded conformations.
Novel cDNAs encoding salivary proteins from the malaria vector Anopheles gambiae
Authors: Lanfrancotti Alessandra , Lombardo Fabrizio , Santolamazza Federica , Veneri Massimiliano , Castrignanò Tiziana , Coluzzi Mario , Arcà Bruno
Published: October 2002

Several genes encoding salivary components of the mosquito Anopheles gambiae were identified using a selective trapping approach. Among these, five corresponded to genes expressed specifically in female glands and their role may possibly be linked to blood‐feeding. Our collection included a fourth member of the D7 protein family and two polypeptides that showed weak similarity to anti‐coagulants from distantly related species. Moreover, we identified two additional members of a novel group of proteins that we named glandins. The isolation of tissue‐specific genes represents a first step toward a deeper molecular analysis of mosquito salivary secretions.
cDNA macroarray analysis of gene expression in synoviocytes stimulated with TNFα
Authors: Sugiyama Tomoyasu , Ishii Shizuko , Yamamoto Jun-ichi , Irie Ryotaro , Saito Kaoru , Otuki Tetsuji , Wakamatsu Ai , Suzuki Yuzuru , Hio Yuri , Ota Toshio , Nishikawa Tetsuo , Sugano Sumio , Masuho Yasuhiko , Isogai Takao
Published: October 2002

Gene expression of synoviocytes stimulated with tumor necrosis factor‐α (TNFα) was studied by macroarray analysis to elucidate the cellular response and identify new biological functions of known and unknown genes. 10 035 cDNA clones were used to make cDNA macroarrays of representative genes. Synoviocytes expressed large amounts of fibronectin and collagen mRNA. Statistical analysis of the macroarray data revealed 26 genes, including six new genes, which underwent significant alteration of gene expression in response to TNFα stimulation. These findings suggest that the synoviocyte response to TNFα stimulation forms the basis of development of various aspects of the pathophysiology of rheumatoid arthritis.
Identification of a Baeyer–Villiger monooxygenase sequence motif
Authors: Fraaije Marco W. , Kamerbeek Nanne M. , van Berkel Willem J.H. , Janssen Dick B.
Published: October 2002

Baeyer–Villiger monooxygenases (BVMOs) form a distinct class of flavoproteins that catalyze the insertion of an oxygen atom in a C–C bond using dioxygen and NAD(P)H. Using newly characterized BVMO sequences, we have uncovered a BVMO‐identifying sequence motif: FXGXXXHXXXW(P/D). Studies with site‐directed mutants of 4‐hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB suggest that this fingerprint sequence is critically involved in catalysis. Further sequence analysis showed that the BVMOs belong to a novel superfamily that comprises three known classes of FAD‐dependent monooxygenases: the so‐called flavin‐containing monooxygenases (FMOs), the N‐hydroxylating monooxygenases (NMOs), and the BVMOs. Interestingly, FMOs contain an almost identical sequence motif when compared to the BVMO sequences: FXGXXXHXXX(Y/F). Using these novel amino acid sequence fingerprints, BVMOs and FMOs can be readily identified in the protein sequence databank.
Role of calmodulin in the modulation of the MAPK signalling pathway and the transactivation of epidermal growth factor receptor mediated by PKC
Authors: Tebar Francesc , Lladó Anna , Enrich Carlos
Published: October 2002

We have recently shown that calmodulin (CaM) regulates the trafficking of epidermal growth factor receptor (EGFR) as well as the mitogen‐activated protein kinase (MAPK) signalling pathway. However, the overall regulation of the MAPK pathway is achieved through a complex interplay of other several upstream effectors including G‐proteins, EGF, EGFR, protein kinase C (PKC), phosphatidylinositol‐3‐kinase and CaM. In order to understand the role of CaM in the PKC‐mediated transactivation of EGFR we have analysed the effect of a CaM antagonist, N‐(4‐aminobutyl)‐5‐chloro‐2‐naphthalenesulfonamide, on the 12‐O‐tetradecanoylphorbol‐13‐acetate‐mediated activation of EGFR and the subsequent MAPK activation. The results show that CaM interferes with MAPK activation and the transactivation of EGFR mediated by PKC.
Design of a chimeric promoter induced by pro‐inflammatory mediators in articular chondrocytes
Authors: Meynier de Salinelles Véronique , Berenbaum Francis , Jacques Claire , Salvat Colette , Olivier Jean-Luc , Béréziat Gilbert , Raymondjean Michel , Massaad Charbel
Published: October 2002

We have designed a chimeric promoter that can be stimulated by various pro‐inflammatory mediators and so drive the expression of therapeutic genes under inflammatory conditions. The promoter has two parts, the [−247/+20] fragment of the human type IIA secreted phospholipase A2 gene promoter, which is stimulated by the pro‐inflammatory cytokine interleukin‐1β (IL‐1β), and a double peroxisome proliferator‐activated receptor response element that is activated by some eicosanoids and by non‐steroidal anti‐inflammatory drugs (NSAIDs). Transfection experiments using rabbit articular chondrocytes in primary culture showed that this chimeric promoter produced a low basal activity and was induced by NSAIDs, WY‐14643, IL‐1β, and 15‐deoxy Δ12,14 prostaglandin J2. The latter two compounds stimulated the promoter synergistically.
Crystal structure of human calmodulin‐like protein: insights into its functional role1
Authors: Han Bong-Gyoon , Han Minhua , Sui Haixin , Yaswen Paul , Walian Peter J , Jap Bing K
Published: October 2002

A calmodulin (CaM)‐like protein (hCLP) is expressed in human mammary epithelial cells but appears to be limited to certain epithelial cells such as those found in skin, prostate, breast and cervical tissues. A decrease in the expression of this protein is associated with the occurrence of tumors in breast epithelium. The structure of hCLP determined to 1.5 Å resolution by X‐ray crystallography shows a distinct 30° displacement along the interconnecting central helix, when compared to the highly conserved structure of vertebrate CaM, resulting in a difference in the relative orientation of its two globular domains. Additionally, the electric surface potential landscape at the target protein binding regions on the two globular domains of hCLP is significantly different from those of CaM, indicating that the respective ranges of hCLP and hCaM target proteins do not fully overlap. Observations that hCLP can competitively inhibit CaM activation of target proteins also imply a role for hCLP in which it may also serve as a modulator of CaM activity in the epithelial cells where hCLP is expressed.
Promotion of cathepsin L activity in newt spermatogonial apoptosis induced by prolactin
Authors: Fujimoto Kenta , Yamamoto Takashi , Kitano Takeshi , Abé Shin-Ichi
Published: October 2002

We previously showed that prolactin (PRL) induces apoptosis in newt secondary spermatogonia and indicated that caspase activity is involved in the apoptosis. Since it was recently reported that Z‐VAD‐fmk, a pan‐caspase inhibitor, blocks activity of cysteine cathepsins as well, we examined whether cathepsin is involved in the newt spermatogonial apoptosis. We found cathepsin L activity in the testis that was elevated by PRL in organ culture of testis, while E‐64d, a lysosomal cysteine protease inhibitor, and Z‐VAD‐fmk suppressed it and chromosomal condensation. These results suggest that cathepsin L activity play a pivotal role in PRL‐induced spermatogonial apoptosis.
ERG6andPDR5regulate small lipophilic drug accumulation in yeast cells via distinct mechanisms
Authors: Emter Roger , Heese-Peck Antje , Kralli Anastasia
Published: October 2002

Diagnosis and circumvention of multi‐drug resistance requires an understanding of the underlying cellular mechanisms. In the model organismSaccharomyces cerevisiae, deletions ofPDR5orERG6increase sensitivity to many small lipophilic drugs. Pdr5p is a plasma membrane ATP‐binding cassette transporter that actively exports drugs, thereby lowering their intracellular levels. The mechanism by whichERG6, an enzyme in sterol biosynthesis, affects drug accumulation is less clear. We show here thatERG6limits the rate of passive drug diffusion across the membrane, without affecting Pdr5p‐mediated drug export. Consistent with their action by distinct mechanisms,PDR5andERG6effects on drug accumulation are additive.
Direct interaction of Frizzled‐1, ‐2, ‐4, and ‐7 with PDZ domains of PSD‐95
Authors: Hering Heike , Sheng Morgan
Published: October 2002

In Drosophila, the frizzled gene plays a critical role in the establishment of tissue polarity, but the function of the Frizzled family of proteins in mammals is largely unknown. Recent evidence suggested that Frizzleds are receptors for the Wnt family of secreted glycoproteins which are involved in cell fate determination. However, it is unclear how Frizzled receptors transduce Wnt signals to intracellular signaling components. Here we show that the mouse Frizzled‐1, ‐2, ‐4 and ‐7 can bind to proteins of the PSD‐95 family, which are implicated in the assembly and localization of multiprotein signaling complexes in the brain. Moreover, PSD‐95 can form a ternary complex with Frizzled‐2 and the adenomatous polyposis coli protein, a negative regulator of Wnt signaling, suggesting that members of the PSD‐95 family may serve to recruit intracellular signaling molecules of the Wnt/Frizzled pathway into the vicinity of the receptor.
Direct modulation of volume‐regulated anion channels by Ca2+ chelating agents
Authors: Lemonnier L , Vitko Y , Shuba Y.M , Vanden Abeele F , Prevarskaya N , Skryma R
Published: October 2002

Ca2+ chelating agents are widely used in biological research for Ca2+ buffering. Here we report that BAPTA, EDTA and HEDTA produce fast, reversible, voltage‐dependent inhibition of swelling‐activated Cl− current (I
Cl,swell) in LNCaP prostate cancer epithelial cells that is unrelated to their Ca2+ binding. BAPTA was the most effective (maximal blockade 67%, IC50=70 μM, at +100 mV) followed by EDTA and HEDTA. I
Cl,swell blockade by EDTA was pH‐dependent. BAPTA blocked I
Cl,swell also in other cell types. We conclude that Ca2+ chelating agents block I
Cl,swell by acting directly on the underlying channel, and that the negative charge of the free chelator form is critical for the blockade.
Protein kinase A balances the growth factor‐induced Ras/ERK signaling
Authors: Pursiheimo Juha-Pekka , Kieksi Anni , Jalkanen Markku , Salmivirta Markku
Published: October 2002

Protein kinase A (PKA) has been proposed to regulate the signal transduction through the Ras/extracellular‐regulated kinase (ERK) pathway. Here we demonstrate that when the PKA activity was inhibited prior to growth factor stimulus the signal flow through the Ras/ERK pathway was significantly increased. Furthermore, the data indicated that this PKA‐mediated regulation was simultaneously targeted to the upstream kinase Raf‐1 and to the ERK‐specific phosphatase mitogen‐activated protein kinase phosphatase‐1 (MKP‐1). Moreover, our data suggested that the level of PKA activity determined the transcription rate of mkp‐1 gene, whereas the Ras/ERK signal was required to protect the MKP‐1 protein against degradation. These results point to a tight regulatory relationship between PKA and the growth factor signaling, and further suggest an important role for basal PKA activity in such regulation. We propose that PKA adjusts the activity of the Ras/ERK pathway and maintains it within a physiologically appropriate level.
A role for calcium in sphingosine 1‐phosphate‐induced phospholipase D activity in C2C12 myoblasts
Authors: Meacci Elisabetta , Becciolini Laura , Nuti Francesca , Donati Chiara , Cencetti Francesca , Farnararo Marta , Bruni Paola
Published: October 2002

Receptor‐regulated phospholipase D (PLD) is a key signaling pathway implicated in the control of fundamental biological processes. Here evidence is presented that in addition to protein kinase C (PKC) and Rho GTPases, Ca2+ response evoked by sphingosine 1‐phosphate (S1P) also participates to the enzyme regulation. Ca2+ was found critical for PKCα‐mediated PLD activation. Moreover, S1P‐induced PLD activity resulted diminished by calmodulin inhibitors such as W‐7 and CGS9343B implicating its involvement in the process. A plausible candidate for Ca2+‐dependent PLD regulation by S1P was represented by calcineurin, in view of the observed reduction of the stimulatory effect by cyclosporin A. In contrast, monomeric GTP‐binding protein Ral was translocated to membranes by S1P in a Ca2+‐independent manner, ruling out its possible role in agonist‐mediated regulation of PLD.
Phosphatidylserine peroxidation/externalization during staurosporine‐induced apoptosis in HL‐60 cells
Authors: Matsura Tatsuya , Serinkan Behice F , Jiang Jianfei , Kagan Valerian E
Published: October 2002

Although oxidative stress is commonly associated with apoptosis, its specific role in the execution of the apoptotic program has yet to be described. We hypothesized that catalytic redox interactions between negatively charged phosphatidylserine (PS) and positively charged cytochrome c released into the cytosol, along with the production of reactive oxygen species (ROS), results in pronounced oxidation and externalization of PS, and subsequent recognition of apoptotic cells by macrophages. By using staurosporine, a protein kinase inhibitor that does not act as a prooxidant, we were able to induce apoptosis in HL‐60 cells without triggering the confounding effects of non‐specific oxidation reactions. Through this approach, we demonstrated for the first time that PS underwent a statistically significant and pronounced oxidation at an early stage (2 h) of non‐oxidant‐induced apoptosis while the most abundant phospholipid, phosphatidylcholine, did not. Glutathione (GSH), the most abundant cytosolic thiol, also remained unoxidized at this time point. Furthermore, PS oxidation and the appearance of cytochrome c in the cytosol were concurrent; PS externalization was followed by phagocytosis of apoptotic cells. These findings are compatible with our proposed roles for oxidative PS‐dependent signaling during apoptosis and phagocytosis.
BE‐18591 as a new H+/Cl− symport ionophore that inhibits immunoproliferation and gastritis
Authors: Tanigaki Keiji , Sato Tomohiko , Tanaka Yasufumi , Ochi Takahiro , Nishikawa Asako , Nagai Kazuo , Kawashima Hiroyuki , Ohkuma Shoji
Published: October 2002

In our previous papers [e.g. Sato et al., J. Biol. Chem. 273 (1998) 21455–21462], we have shown that prodigiosins can uncouple various H+‐ATPases through their H+/Cl− symport activity. BE‐18591 is an enamine of 4‐methoxy‐2,2′‐bipyrrole‐5‐carboxyaldehyde (tambjamine group antibiotics) which resembles the prodigiosins. We found that BE‐18591 was a new group of antibiotics that uncouples various H+‐ATPases: it inhibited proton pump activities with IC50s of about 1–2 nM (about 20 pmol/mg protein) for submitochondrial particles as well as gastric vesicles and of 230 nM (about 230 pmol/mg protein) for lysosomes, but it had little effect on their ATP hydrolyses (up to 10 μM), a property of H+/Cl− symport activity. At low concentrations (<1 μM), BE‐18591 inhibited immunoproliferation, the IC50 of lipopolysaccharide‐stimulated mouse splenocytes was 38 nM, that of Concanavalin A‐stimulated cells was 230 nM. Gastritis of rabbits was also inhibited. At higher concentrations (>1 μM), BE‐18591 induced neurite outgrowth (15% induction in 48 h at 4 μM), inhibited bone resorption (∼35% in 48 h at 10 μM) and caused cell death (∼30% in 48 h at 4 μM) but with little apoptosis.
An ovarian follicular epithelium protein of the silkworm (Bombyx mori) that associates with the vitelline membrane and contributes to the structural integrity of the follicle
Authors: Kendirgi Frederic , Swevers Luc , Iatrou Kostas
Published: October 2002

We have cloned and functionally characterized a novel protein, BmVMP30, which is synthesized by the cells of the follicular epithelium of the ovarian follicles of the domesticated silkworm Bombyx mori, secreted from them and associated with the vitelline membrane. BmVMP30 is a 30 kDa protein that bears limited structural features reminiscent of other insect vitelline membrane proteins. Although BmVMP30 does not share pronounced similarities or signature motifs with other reported proteins, its temporal and spatial expression and its behavior throughout oogenesis suggest that it is a novel member of the insect vitelline membrane protein family. The protein is expressed exclusively in the cells of the follicular epithelium during stages −15 to −1 of vitellogenesis, secreted from them and, ultimately, localized at the junction between the oocyte and the eggshell, where the vitelline membrane is located. Treatment of follicles with an antisense oligonucleotide that encompasses the translation initiation codon results in the production of an N‐terminally truncated protein and disruption of the integrity of the follicular epithelium. Antisense oligonucleotide treatment, however, has no effect on the implementation of the developmental program that directs the autonomous progression of ovarian follicles through the last stages of vitellogenesis and choriogenesis.
Identification of functional p53‐binding motifs in the mouse wig‐1 promoter
Authors: Wilhelm Margareta T , Méndez-Vidal Cristina , Wiman Klas G
Published: October 2002

We previously identified wig‐1 as a p53‐induced mouse gene that encodes a nuclear zinc finger protein with unknown function. To investigate whether wig‐1 is a direct target of p53‐dependent transactivation, a DNA fragment corresponding to the promoter region was cloned and sequenced. Three regions containing consensus p53‐binding sites were identified. Two p53‐binding motifs formed DNA–protein complexes with p53 and were able to drive p53‐dependent transcription in a luciferase reporter assay. Our results demonstrate that wig‐1 is a direct target of p53‐mediated transcriptional transactivation.
Cmk2, a novel serine/threonine kinase in fission yeast
Authors: Alemany Vicenç , Sanchez-Piris Maribel , Bachs Oriol , Aligue Rosa
Published: October 2002

The cmk2 gene of Schizosaccharomyces pombe encodes a 504 amino acid protein kinase with sequence homology with the calmodulin‐dependent protein kinase family. The cmk2
+ gene is not essential for cell viability but overexpression of cmk2
+ blocks the cell cycle at G2 phase and this inhibition is cdc2‐dependent. The Cmk2 is a cytoplasmic protein expressed in a cell cycle‐dependent manner, peaking at the G1/S boundary. Overexpression of Cmk2 suppresses fission yeast DNA replication checkpoint defects but not DNA damage checkpoint defects, suggesting that the G2 cell cycle arrest mediated by high levels of Cmk2 provides sufficient time to correct DNA replication alterations.
Zn2+ site engineering at the oligomeric interface of the dopamine transporter
Authors: Norgaard-Nielsen Kristine , Norregaard Lene , Hastrup Hanne , Javitch Jonathan A , Gether Ulrik
Published: October 2002

Increasing evidence suggests that Na+/Cl−‐dependent neurotransmitter transporters exist as homo‐oligomeric proteins. However, the functional implication of this oligomerization remains unclear. Here we demonstrate the engineering of a Zn2+ binding site at the predicted dimeric interface of the dopamine transporter (DAT) corresponding to the external end of transmembrane segment 6. Upon binding to this site, which involves a histidine inserted in position 310 (V310H) and the endogenous Cys306 within the same DAT molecule, Zn2+ potently inhibits [3H]dopamine uptake. These data provide indirect evidence that conformational changes critical for the translocation process may occur at the interface between two transporter molecules in the oligomeric structure.
Mechano‐chemical effects of Ca2+ in cross‐linked troponin‐C films
Authors: Bukatina Anna E , Morozov Victor N , Gusev Nikolai B , Sieck Gary C
Published: October 2002

Changes in troponin C (TnC) conformation upon Ca2+ binding forms the basis for regulatory and structural functions of TnC molecules. In the present study, Ca2+‐induced conformational changes in TnC were observed by mechanical measurements. TnC films were prepared by drying or electrospraying TnC solutions, cross‐linked with glutaraldehyde, and isometric tension and stiffness measured as a function of pCa. An increase in Ca2+ from a pCa of 9 to 4 induced large‐scale mechanical changes in the TnC films causing several percent shrinkage of the unloaded films. This shrinkage could be partially assigned to Ca2+ binding to the Ca2+/Mg2+ sites of TnC.