bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of the cardiac voltage‐gated Na+ channel (H1) by the ubiquitin‐protein ligase Nedd4
Authors: Abriel Hugues , Kamynina Elena , Horisberger Jean-Daniel , Staub Olivier
Published: July 2002

The cardiac voltage‐gated Na+ channel H1, involved in the generation of cardiac action potential, contains a C‐terminal PY motif (xPPxY). Since PY motifs are known ligands to WW domains, we investigated their role for H1 regulation and the possible involvement of the WW domain containing ubiquitin‐protein ligase Nedd4, taking advantage of the Xenopus oocyte system. Mutation of the PY motif leads to higher peak currents when compared to wild‐type channel. Moreover, co‐expression of Nedd4 reduced the peak currents, whereas an enzymatically inactive Nedd4 mutant increased them, likely by competing with endogenous Nedd4. The effect of Nedd4 was not observed in the PY motif mutated channel or in the skeletal muscle voltage‐gated Na+ channel, which lacks a PY motif. We conclude that H1 may be regulated by Nedd4 depending on WW–PY interaction, and on an active ubiquitination site.
LOX‐1 mediates lysophosphatidylcholine‐induced oxidized LDL uptake in smooth muscle cells
Authors: Aoyama Takuma , Chen Mingyi , Fujiwara Hisayoshi , Masaki Tomoh , Sawamura Tatsuya
Published: July 2002

A novel receptor for oxidized low‐density lipoprotein (OxLDL), lectin‐like OxLDL receptor (LOX‐1), was cloned from endothelial cells. Since OxLDL is taken up by vascular smooth muscle cells (VSMC) in atheroma, we analyzed the inducible expression of LOX‐1 in VSMC in the present study. Incubation of cultured bovine VSMC with lysophosphatidylcholine (LPC), an atherogenic component of OxLDL, increased the level of mRNA for LOX‐1 in a dose‐ and time‐dependent manner. Since LPC did not significantly change the half‐life of LOX‐1 mRNA, the induction seemed to occur at the transcriptional level. The induction accompanied an increase in the protein level of LOX‐1 and activity of OxLDL uptake. Blocking antibody against LOX‐1 significantly suppressed the enhanced uptake of OxLDL. Thus, LOX‐1 is a major receptor for OxLDL in VSMC as in endothelial cells. The enhanced expression of LOX‐1 by LPC suggests that OxLDL and LPC would progressively change the function of VSMC and accelerate atherogenesis in vivo.
Chlorophyll fluorescence quenching in isolated light harvesting complexes induced by zeaxanthin
Authors: Wentworth Mark , Ruban Alexander V. , Horton Peter
Published: July 2002

Non‐photochemical quenching of chlorophyll fluorescence in plants occurs in the light harvesting antenna of photosystem II and is regulated by the xanthophyll cycle. A new in vitro model for this process has been developed. Purified light harvesting complexes above the detergent critical micelle concentration have a stable high fluorescence yield but a rapidly inducible fluorescence quenching occurs upon addition of zeaxanthin. Violaxanthin was without effect, lutein and antheraxanthin induced a marginal response, whereas the violaxanthin analogue, auroxanthin, induced strong quenching. Quenching was not caused by aggregation of the complexes but was accompanied by a spectral broadening and red shift, indicating a zeaxanthin‐dependent alteration in the chlorophyll environment.
Expression of a constitutively active form of phosphatidylinositol 3‐kinase inhibits the induction of nitric oxide synthase in human astrocytes
Authors: Pahan Kalipada , Liu Xiaojuan , Wood Charles , Raymond John R.
Published: July 2002

The present study underlines the importance of phosphatidylinositol 3‐kinase (PI 3‐kinase) in attenuating the induction of nitric oxide synthase (iNOS) in human astrocytes. Proinflammatory cytokines induced the production of nitric oxide (NO) and the expression of iNOS in human U373MG astrocytoma cells and primary astrocytes. Expression of a catalytically active p110 subunit (p110*) of PI 3‐kinase but not that of a kinase‐deficient mutant of p110 (p110‐kd) induced an increase in PI 3‐kinase activity and inhibited cytokine‐induced production of NO and expression of iNOS. However, expression of p110* had no effect on the activation of NF‐kB, suggesting that p110* inhibits the expression of iNOS without inhibiting the activation of NF‐kB.
hKCNMB3 and hKCNMB4, cloning and characterization of two members of the large‐conductance calcium‐activated potassium channel β subunit family
Authors: Behrens R. , Nolting A. , Reimann F. , Schwarz M. , Waldschütz R. , Pongs O.
Published: July 2002

We cloned two β subunits of large‐conductance calcium‐activated potassium (BK) channels, hKCNMB3 (BKβ1) and hKCNMB4 (BKβ4). Profiling mRNA expression showed that hKCNMB3 expression is enriched in testis and hKCNMB4 expression is very prominent in brain. We coexpressed BK channel α (BKα) and BKβ4 subunits in vitro in CHO cells. We compared BKα/β4 mediated currents with those of smooth muscle BKα/β1 channels. BKβ4 slowed activation kinetics more significantly, led to a steeper apparent calcium sensitivity, and shifted the voltage range of BK current activation to more negative potentials than BKβ1. BKα/β4 channels were not blocked by 100 nM charybdotoxin or iberiotoxin, and were activated by 17β‐estradiol.
Priming of PC12 cells for semiquantitative microinjection studies involving Ras
Authors: Schmidt Gudula , Wittinghofer Alfred
Published: July 2002

Nerve growth factor and activated Ras can induce differentiation of rat pheochromocytoma cells (PC12 cells) [Greene and Tischler (1976) Proc. Natl. Acad. Sci. USA 73, 2424–2428] from a chromaffin cell‐like morphology into one that resembles sympathetic neurones. We developed a special treatment of PC12 cells which apparently synchronises these cells such that they are more useful for semi‐quantitative microinjection studies for signal transduction pathways. This treatment leads to a faster and more reproducible differentiation which faithfully reproduces the involvement of Ras in the process and allows a comparison of the biological activity of different Ras mutants. It shows that G12V and Q61L oncogenic mutants are not equally potent in inducing differentiation. Partial loss‐of‐function mutations T35S, E37G and Y40C are inactive and even a triple combination of these does not restore full biological activity.
Identification of a second site compensatory mutation in the Fe‐protein that allows diazotrophic growth of Azotobacter vinelandii UW97
Authors: Lei Shi , Pulakat Lakshmidevi , Suh Man-Hee , Gavini Narasaiah
Published: July 2002

Azotobacter vinelandii UW97 is defective in nitrogen fixation due to a replacement of serine at position 44 by phenylalanine in the Fe‐protein [Pulakat, L., Hausman, B.S., Lei, S. and Gavini, N. (1996) J. Biol. Chem. 271, 1884–1889]. Serine residue 44 is located in a conserved domain that links the nucleotide binding site and the MoFe‐protein docking surface of the Fe‐protein. Therefore, it is possible that the loss of function by A. vinelandii UW97‐Fe‐protein may be caused by global conformational disruption or disruption of the conformational change upon MgATP binding. To determine whether it is possible to generate a functional nitrogenase complex via a compensating second site mutation(s) in the Fe‐protein, we have attempted to isolate genetic revertants of A. vinelandii UW97 that can grow on nitrogen‐free medium. One such revertant, designated A vinelandii BG9, encoded a Fe‐protein that retained the Ser44Phe mutation and also had a second mutation that caused the replacement of a lysine at position 170 by glutamic acid. Lysine 170 is highly conserved and is located in a conserved region of the Fe‐protein. This region is implicated in stabilizing the MgATP‐induced conformation of the Fe‐protein and in docking to the MoFe‐protein. Further complementation analysis showed that the Fe‐protein mutant that retained serine 44 but contained the substitution of lysine at position 170 by glutamic acid was also non‐functional. Thus, neither Ser44Phe nor Lys170Glu mutants of Fe‐protein were functional; however, the Fe‐protein in A. vinelandii BG9 that contained both substitutions could support diazotrophic growth on the strain.
Single‐channel analysis of a ClC‐2‐like chloride conductance in cultured rat cortical astrocytes
Authors: Nobile M. , Pusch M. , Rapisarda C. , Ferroni S.
Published: July 2002

The single‐channel behavior of the hyperpolarization‐activated, ClC‐2‐like inwardly rectifying Cl− current (I
Clh), induced by long‐term dibutyryl‐cyclic‐AMP‐treated cultured cortical rat astrocytes, was analyzed with the patch‐clamp technique. In outside‐out patches in symmetrical 144 mM Cl− solutions, openings of hyperpolarization‐activated small‐conductance Cl− channels revealed burst activity of two equidistant conductance levels of 3 and 6 pS. The unitary openings displayed slow activation kinetics. The probabilities of the closed and conducting states were consistent with a double‐barrelled structure of the channel protein. These results suggest that the astrocytic ClC‐2‐like Cl− current I
Clh is mediated by a small‐conductance Cl− channel, which has the same structural motif as the Cl− channel prototype ClC‐0.
The growth suppressing gas1 product is a GPI‐linked protein
Authors: Stebel Marco , Vatta Paolo , Ruaro Maria Elisabetta , Del Sal Giannino , Parton Robert G. , Schneider Claudio
Published: July 2002

Growth arrest specific (gas) 1 gene product is expressed in non‐transformed fibroblasts in response to stimuli driving cells into Go phase. Gas1 has been demonstrated to inhibit cell proliferation when over‐expressed in proliferating fibroblasts. This activity depends on a function of the p53 protein independent of its transactivating ability. To better define the pathway leading from Gas1, which is located on the plasma membrane, to p53, we have undertaken a detailed characterization of its topology. We demonstrate that the protein undergoes cotranslational modifications in the endoplasmic reticulum, consisting of signal peptide cleavage, N‐linked glycosylation and glycosyl‐phosphatidylinositol anchor addition. Immunoelectron microscopy shows that, in its mature form, Gas1 is randomly distributed over the outer leaflet of the plasma membrane and that upon antibody‐induced clustering it relocalizes to caveolae.
Lovastatin arrests CHO cells between the origin decision point and the restriction point
Authors: Wu Jia-Rui , Gilbert David M.
Published: July 2002

Asynchronously growing Chinese hamster ovary (CHO) cells treated with the pro‐drug, β‐lactone ring form of lovastatin were arrested in G1‐phase. Subsequent removal of lovastatin resulted in the synchronous entry of cells into S‐phase regardless of the presence of mevalonic acid. Lovastatin‐arrested cells contained hypophosphorylated retinoblastoma protein (Rb) and required serum mitogens to enter S‐phase after lovastatin removal, indicating that cell‐cycle arrest is prior to the restriction point (R‐point). However, in contrast to quiescent cells, intact nuclei prepared from lovastatin‐arrested cells were competent for DNA replication when introduced into Xenopus egg extracts. Initiation of replication by Xenopus egg cytosol took place specifically within the dihydrofolate reductase (DHFR) origin locus, demonstrating that cells were arrested after the origin decision point (ODP). We conclude that the β‐lactone ring form of lovastatin is an effective reagent with which to synchronize CHO cells between the ODP and R‐point, without resulting in the withdrawal of cells from the cell‐cycle into a quiescent state.
HSP70 and ribosomal protein L2: novel 5S rRNA binding proteins in Escherichia coli
Authors: Okada Saori , Okada Tetsuji , Aimi Tadanori , Morinaga Tsutomu , Itoh Takuzi
Published: July 2002

A Northwestern analysis of Escherichia coli total proteins with radiolabeled 5S rRNA identified two novel 5S rRNA interacting proteins, a 70 kDa and a 37 kDa protein, and three ribosomal proteins reported on already. The N‐terminal sequencing of the 70 kDa protein separated by SDS–PAGE from the high‐salt‐washed fraction of crude ribosome led to the discovery of a polypeptide identical in its first 10 amino acid residues to E. coli heat shock protein 70. The N‐terminal eight amino acid sequence of the 37 kDa protein extracted from the high‐salt‐washed ribosome is identical to that of the ribosomal protein L2. In addition, the interaction of these proteins with 5S rRNA has been confirmed with gel mobility shift assays.
β‐Agonists regulate Na,K‐ATPase via novel MAPK/ERK and rapamycin‐sensitive pathways
Authors: Pesce Liuska , Guerrero Carmen , Comellas Alejandro , Ridge Karen M. , Sznajder Jacob I.
Published: July 2002

We studied whether the β‐adrenergic agonist, isoproterenol (ISO), regulates Na,K‐ATPase in alveolar epithelial cells (AEC) via a mitogen‐activated protein kinase (MAPK)/extracellular signaling related kinase (ERK) dependent pathway. ISO increased ERK activity in AEC by 10 min via a β‐adrenergic receptor, protein kinase A (PKA)‐dependent mechanism. Activation of the MAPK pathway by ISO, resulted in increased Na,K‐ATPase β1 and α1 subunit protein abundance in whole cell lysates, which resulted in functional Na,K‐ATPases at the basolateral membranes. ISO did not change the α1 or β1 mRNA steady state levels, but rapamycin, the inhibitor of the mammalian target of rapamycin, also blocked the ISO‐mediated increase in Na,K‐ATPase total protein abundance, suggesting a posttranscriptional regulation. We conclude that ISO, regulates the Na,K‐ATPase in AEC via PKA, ERK and rapamycin‐sensitive mechanisms.
Ca2+ sensors of L‐type Ca2+ channel
Authors: Romanin Christoph , Gamsjaeger Roland , Kahr Heike , Schaufler Daniela , Carlson Olga , Abernethy Darrell R. , Soldatov Nikolai M.
Published: July 2002

Ca2+‐induced inactivation of L‐type Ca2+ is differentially mediated by two C‐terminal motifs of the α1C subunit, L (1572–1587) and K (1599–1651) implicated for calmodulin binding. We found that motif L is composed of a highly selective Ca2+ sensor and an adjacent Ca2+‐independent tethering site for calmodulin. The Ca2+ sensor contributes to higher Ca2+ sensitivity of the motif L complex with calmodulin. Since only combined mutation of both sites removes Ca2+‐dependent current decay, the two‐site modulation by Ca2+ and calmodulin may underlie Ca2+‐induced inactivation of the channel.
Site‐directed mutagenesis of K396R of the 65 kDa glutamic acid decarboxylase active site obliterates enzyme activity but not antibody binding
Authors: Hampe Christiane S. , Hammerle Lisa P. , Falorni Alberto , Robertson John , Lernmark Åke
Published: July 2002

The role of K396 in the enzymatic catalysis and the antigenicity of the 65 kDa isoform of glutamate decarboxylase (GAD65) was analyzed using the K396R GAD65 mutant. GAD65 is a major autoantigen in Type 1 diabetes and autoantibodies directed to GAD65 are widely used markers for this disease. We found that (1) recombinant human GAD65 is fully enzymatically active; (2) the K396R mutation abolished GAD65 activity; and (3) the K396R mutant retained full antigenicity to GAD65 autoantibodies in serum from Type 1 diabetes patients, but not to polyclonal antibodies raised to the catalytic domain.
Agonist‐stimulated internalisation of the ligand‐gated ion channel P2X1 in rat vas deferens
Authors: Ennion Steven J , Evans Richard J
Published: July 2002

Using cell surface biotinylation and Western blotting, we investigated the extent to which native P2X1 receptors in rat vas deferens are internalised after exposure to agonist. Exposure to 100 μM α,β‐meATP 30 min prior and during a 10 min biotinylation period resulted in a ∼50% reduction in the amount of biotinylated P2X1 receptor indicating that activation of the receptor by agonist induces receptor internalisation. Furthermore, biotinylation under saturating conditions suggests that once internalised, a rapid recycling of P2X1 receptor back to the cell surface occurs. The physiological implications of these mechanisms in terms of receptor function are discussed.
Conserved role for 14‐3‐3ϵ downstream of type I TGFβ receptors
Authors: McGonigle Sharon , Beall Melissa J. , Feeney Erika L. , Pearce Edward J.
Published: July 2002

Schistosoma mansoni receptor kinase‐1 (SmRK1) is a divergent type I transforming growth factor β (TGFβ) receptor on the surface of adult parasites. Using the intracellular domain of SmRK1 as bait in a yeast two‐hybrid screen we identified an interaction with S. mansoni 14‐3‐3ϵ. The interaction which is phosphorylation‐dependent is not specific to schistosomes since 14‐3‐3ϵ also binds to TβRI, the human type I TGFβ receptor. 14‐3‐3ϵ enhances TGFβ‐mediated signaling by TβRI and is the first TβRI‐interacting non‐Smad protein identified that positively regulates this receptor. The interaction of 14‐3‐3ϵ with schistosome and human TβRI suggests a conserved, but previously unappreciated, role for this protein in TGFβ signaling pathways.
Localization of phospholipase C‐γ1 signaling in caveolae: importance in EGF‐induced phosphoinositide hydrolysis but not in tyrosine phosphorylation
Authors: Jang Il-Ho , Kim Jae Ho , Lee Byoung Dae , Bae Sun Sik , Park Myung Hwan , Suh Pann-Ghill , Ryu Sung Ho
Published: July 2002

Upon epidermal growth factor treatment, phospholipase C‐γ1 (PLC‐γ1) translocates from cytosol to membrane where it is phosphorylated at tyrosine residues. Caveolae are small plasma membrane invaginations whose structural protein is caveolin. In this study, we show that the translocation of PLC‐γ1 and its tyrosine phosphorylation are localized in caveolae by caveolin‐enriched low‐density membrane (CM) preparation and immunostaining of cells. Pretreatment of cells with methyl‐β‐cyclodextrin (MβCD), a chemical disrupting caveolae structure, inhibits the translocation of PLC‐γ1 to CM as well as phosphatidylinositol (PtdIns) turnover. However, MβCD shows no effect on tyrosine phosphorylation level of PLC‐γ1. Our findings suggest that, for proper signaling, PLC‐γ1 phosphorylation has to occur at PtdInsP2‐enriched sites.
The BPS domain of Grb10 inhibits the catalytic activity of the insulin and IGF1 receptors
Authors: Stein Evan G. , Gustafson Thomas A. , Hubbard Stevan R.
Published: July 2002

Grb7, Grb10 and Grb14 comprise a family of adaptor proteins that interact with numerous receptor tyrosine kinases upon receptor activation. Between the pleckstrin homology (PH) domain and the Src homology 2 (SH2) domain of these proteins is a region of approximately 50 residues known as the BPS (between PH and SH2) domain. Here we show, using purified recombinant proteins, that the BPS domain of Grb10 directly inhibits substrate phosphorylation by the activated tyrosine kinase domains of the insulin receptor and the insulin‐like growth factor 1 (IGF1) receptor. Although inhibition by the BPS domain is dependent on tyrosine phosphorylation of the kinase activation loop, peptide competition experiments indicate that the BPS domain does not bind directly to phosphotyrosine. These studies provide a molecular mechanism by which Grb10 functions as a negative regulator of insulin‐ and/or IGF1‐mediated signaling.
Molecular structure of the glibenclamide binding site of the β‐cell KATP channel
Authors: Mikhailov Michael V , Mikhailova Ellina A , Ashcroft Stephen J.H
Published: July 2002

We have investigated the structure of the glibenclamide binding site of pancreatic β‐cell ATP‐sensitive potassium (KATP) channels. KATP channels are a complex of four pore‐forming Kir6.2 subunits and four sulfonylurea receptor (SUR1) subunits. SUR1 (ABCC8) belongs to the ATP binding cassette family of proteins and has two nucleotide binding domains (NBD1 and NBD2) and 17 putative transmembrane (TM) sequences. Co‐expression in a baculovirus expression system of two parts of SUR1 between NBD1 and TM12 leads to restoration of glibenclamide binding activity, whereas expression of either individual N‐ or C‐terminal part alone gave no glibenclamide binding activity, confirming a bivalent structure of the glibenclamide binding site. By using N‐terminally truncated recombinant proteins we have shown that CL3 – the cytosolic loop between TM5 and TM6 – plays a key role in formation of the N‐terminal component of the glibenclamide binding site. Analysis of deletion variants of the C‐terminal part of SUR1 showed that CL8 – the cytosolic loop between TM15 and TM16 – is the only determinant for the C‐terminal component of the glibenclamide binding site. We suggest that in SUR1 in the native KATP channel close proximity of CL3 and CL8 leads to formation of the glibenclamide binding site.
Cell surface‐associated chondroitin sulfate proteoglycans bind contact phase factor H‐kininogen
Authors: Renné Thomas , Müller-Esterl Werner
Published: July 2002

The kinin system has been recognized as a locally operating hormone system of cardiovascular cells, however, the molecular mechanisms regulating circumscribed kinin release on cell surfaces are not fully understood. In particular, the principal cell docking sites for the kinin precursor, high molecular weight kininogen (HK), are not fully explored. Here we demonstrate by enzymatic digestion, recombinant overexpression, and affinity cross‐linking studies that cell surface chondroitin sulfate (CS) chains of proteoglycans (PGs) serve as major HK binding sites on platelet, fibroblast, liver, and endothelial kidney cells. In this way, CS‐type PGs may contribute to a local accumulation of kinin precursors on cell surfaces and modulate circumscribed release of short‐lived kinin hormones at or next to their site of action.
The importance of the C‐terminal region and Cys residues for the membrane association of the NADPH:protochlorophyllide oxidoreductase in pea
Authors: Aronsson Henrik , Sundqvist Christer , Timko Michael P. , Dahlin Clas
Published: July 2002

In vitro chloroplast import reactions and thylakoid association reactions have been performed with a series of C‐terminal deletions and Cys‐to‐Ser substitution mutants of the pea NADPH:protochlorophyllide oxidoreductase (POR; EC 1.6.99). C‐terminal deletions of the precursor POR (Δ362–400, Δ338–400, Δ315–400 and Δ300–400) were efficiently translocated across the chloroplast envelope. However, except the Δ396–400 mutant, no C‐terminal deletion mutants or Cys‐to‐Ser substitution (Cys119, Cys281 and Cys309) mutants resisted post‐treatment with thermolysin after the thylakoid association reactions. This suggests that these mutants were unable to properly associate to the thylakoids due to changes of the protein conformation of POR.
Expression of human TRPC genes in the megakaryocytic cell lines MEG01, DAMI and HEL
Authors: Berg Lutz-Peter , Shamsher Monee K , El-Daher Samer S , Kakkar Vijay V , Authi Kalwant S
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Redox‐regulated expression of glycolytic enzymes in resting and proliferating rat thymocytes
Authors: Hamm-Künzelmann Brigitte , Schäfer Doris , Weigert Cora , Brand Karl
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Vacuolar‐type H+‐ATPase in mouse bladder epithelium is responsible for urinary acidification
Authors: Tomochika Ken-ichi , Shinoda Sumio , Kumon Hiromi , Mori Masaharu , Moriyama Yoshinori , Futai Masamitsu
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Simultaneous induction of stimulatory and inhibitory signals by PDGF
Authors: Heldin Carl-Henrik
Published: July 2002

Platelet‐derived growth factor (PDGF) exerts its effects on cells via binding to structurally similar α‐ and β‐tyrosine kinase receptors. Ligand binding induces receptor dimerization and autophosphorylation which allows docking of SH2 domain containing signal transduction molecules. At least 10 different SH2 domain molecules bind in a specific manner to 11 identified autophosphorylated tyrosine residues in the PDGF β‐receptor, thereby initiating signaling pathways leading to cell growth and motility. Available information indicates that there is considerable cross‐talk between different signaling pathways, and that stimulatory and inhibitory signals often are initiated in parallel.
Leptin is a four‐helix bundle: secondary structure by NMR
Authors: Kline Allen D , Becker Gerald W , Churgay Lisa M , Landen Bryan E , Martin Debra K , Muth William L , Rathnachalam Radhakrishnan , Richardson John M , Schoner Brigitte , Ulmer Maverick , Hale John E
Published: July 2002

Leptin is a signaling protein that in its mutant forms has been associated with obesity and Type II diabetes. The lack of sequence similarity has precluded analogies based on structural resemblance to known systems. Backbone NMR signals for mouse leptin (13C/15N ‐labeled) have been assigned and its secondary structure reveals it to be a four‐helix bundle cytokine. Helix lengths and disulfide pattern are in agreement with leptin as a member of the short‐helix cytokine family. A three‐dimensional model was built verifying the mechanical consistency of the identified elements with a short‐helix cytokine core.
Deletion in the Z‐line region of the titin gene in a baby hamster kidney cell line, BHK‐21‐Bi
Authors: Jäckel Monika , Witt Christian , Antonova Olga , Curdt Ingo , Labeit Siegfried , Jockusch Harald
Published: July 2002

The gene for titin, a 4MDa myofibrillar protein, was analysed in golden hamster DNAs from different sources, using human cDNA probes and PCR. In the DNA from the BHK‐21‐Bi subline of baby hamster kidney cells, extended sequences coding for Z‐line associated domains were missing, indicating a deletion that renders titin non‐functional. These sequences were present in the original BHK‐21 line and in hamster DNAs. Our finding shows that, due to the absence of selective pressure on a gene's function, genomic deterioration can occur in a permanent cell line and can lead to a loss of overlapping DNA stretches in both autosomes.
Repression of interleukin‐6 gene expression by 17β‐estradiol:
Authors: Ray Prabir , Ghosh Samir K , Zhang Dong-Hong , Ray Anuradha
Published: July 2002

The cytokine interleukin‐6 (IL‐6), a key mediator of immune and acute phase responses of the liver, has also been implicated in uterine functions. Estrogens are potent repressors of IL‐6 production by uterine stromal cells. In the endometrial adenocarcinoma cell line Ishikawa, phorbol ester‐induced activation of the IL‐6 promoter was inhibited to basal levels by 17β‐estradiol (E2) in a wild‐type receptor‐dependent fashion. Although tamoxifen has been shown to have estrogenic effects on the endometrium, it did not inhibit induction of the IL‐6 promoter. We previously showed that inhibition of IL‐6 gene expression by E2 does not involve high‐affinity binding of the estrogen receptor (ER) to IL‐6 DNA. We now report that the ER can directly interact with the transcription factors NF‐IL6 and NF‐κB and can inhibit their ability to bind DNA which might be the molecular basis for repression of IL‐6 gene expression by estrogens.
Palytoxin effects through interaction with the Na,K‐ATPase in Xenopus oocyte
Authors: Wang Xinyu , Horisberger Jean-Daniel
Published: July 2002

Palytoxin (PTX) is known to bind to Na,K‐ATPase, to inhibit its activity, and to induce cation conductance, but the mechanism of these effects is still poorly understood. In Xenopus oocytes, PTX induced a large cation conductance, an effect that could be prevented or reversed by ouabain for oocytes expressing Xenopus Na,K‐pumps but not with those expressing Bufo Na,K‐pumps. In both cases patch‐clamp experiments demonstrated a 7–8 pS channel in the presence of PTX. A large PTX‐induced conductance could be observed with minimal Na,K‐pump inhibition. From the single PTX‐induced channel and macroscopic whole oocyte conductance, and the number of Na,K‐pumps, we can conclude that PTX‐induced conductance occurs through a direct interaction of PTX with a small number of Na,K‐pumps.
Active oxygen species mediate the solar ultraviolet radiation‐dependent increase in the tumour suppressor protein p53 in human skin fibroblasts
Authors: Vile Glenn F
Published: July 2002

Active oxygen species mediate many of the biological consequences of exposing cultured human skin cells to solar ultraviolet (UV) radiation (290–380 nm). A critical step in the escape from the carcinogenic potential of UV radiation is mediated by the protein p53. P53 activates growth arrest, allowing for DNA repair, and apoptosis, which removes damaged cells. Here I show that p53 in cultured human skin fibroblasts is elevated after treatment with hydrogen peroxide, an oxidant produced in cells during exposure to solar UV radiation. Simulated solar UV radiation increased p53, and agents that scavenge active oxygen species, N‐acetylcysteine, ascorbate and α‐tocopherol, inhibited the increase. The generation of DNA single strand breaks has been proposed to be an important step in the pathway leading to the increase in p53 initiated by a variety of cytotoxic agents. In this study I show that compounds that allow the accumulation of DNA single strand breaks, ara c and hydroxyurea, enhanced the UVC radiation (254 nm)‐dependent increase in p53, but had no effect on the solar UV radiation‐dependent increase. Thus, while DNA single strand breaks are involved in the UVC radiation‐dependent increase in p53, the increase caused by solar UV radiation occurs by an alternative mechanism involving active oxygen species.
Involvement of HNF‐1 in the regulation of phosphoenolpyruvate carboxykinase gene expression in the kidney
Authors: Cassuto Hanoch , Olswang Yael , Livoff Alejandro F , Nechushtan Hovav , Hanson Richard W , Reshef Lea
Published: July 2002

The cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene is differentially expressed in several tissues. A specific set of regulatory elements in the promoter are responsible for the control of PEPCK gene transcription and, in turn, determine its distinct metabolic role in each tissue. DNase I footprinting analysis of the PEPCK promoter, using nuclear proteins from tissues which express the gene for PEPCK, and transient expression assays in renal cell lines have demonstrated that the HNF‐1 recognition motif (P2) in the PEPCK promoter characterizes kidney‐specific expression. This site is required also for the response to acidosis. Since the P2 site is not involved in the expression of the PEPCK gene in the liver, we propose that its critical role in the kidney stems from a combination of abundance of HNF‐1 together with low concentrations of members of the C/EBP family in this tissue.
Immunoliposomes bearing polyethyleneglycol‐coupled Fab′ fragment show prolonged circulation time and high extravasation into targeted solid tumors in vivo
Authors: Maruyama Kazuo , Takahashi Nobuya , Tagawa Toshiaki , Nagaike Kazuhiro , Iwatsuru Motoharu
Published: July 2002

We have developed a new type of long‐circulating immunoliposome (Fab′–PEG immunoliposomes) which is efficiently extravasated into the targeted solid tumor in vivo. Small unilamellar liposomes (100–130 nm in diameter) were prepared from distearoylphosphatidylcholine (DSPC), cholesterol (CHOL) and a dipalmitoylphosphatidylethanolamine derivative of PEG with a terminal maleimidyl group (DPPE‐PEG‐Mal), and conjugated Fab′ fragment of antibody. Inclusion of DPPE‐PEG‐Mal and linkage of the Fab′ fragment instead of intact antibody to PEG terminals allowed the liposomes to evade RES uptake and remain in the circulation for a long time, resulting in enhanced accumulation of the liposomes in the solid tumor. Because of the ability of such Fab′–PEG immunoliposomes to target solid tumors, they appear highly attractive as carriers of not only chemotherapeutic agents, but also of macromolecular drugs.
Clathrin interacts specifically with amphiphysin and is displaced by dynamin1
Authors: McMahon Harvey T , Wigge Patrick , Smith Corrin
Published: July 2002

Amphiphysin is an SH3 domain protein that has been implicated in synaptic vesicle endocytosis. We have recently cloned a second amphiphysin isoform, Amph2 (sequence submitted to GenBank, Y13380). Proteins capable of forming a complex with amphiphysin were isolated from rat brain by using recombinant GST‐Amph2 for binding experiments. As well as interacting with dynamin I, the full‐length protein bound to a weaker 180‐kDa band. Immunoblotting demonstrated this protein to be clathrin. To address whether this is a direct interaction, the clathrin binding to amphiphysin was reconstituted in vitro with purified proteins. The N‐terminal domain of Amph2 is sufficient for clathrin binding. Dynamin, which interacts with the SH3 domain of Amph2, displaces clathrin from the N‐terminus. We propose a model that may explain how clathrin and dynamin are recruited to non‐overlapping sites of the coated pit.
Concomitant and hormonally regulated expression of trp genes in bovine aortic endothelial cells
Authors: Chang Albert S. , Chang Sharon M. , Garcia Reynaldo L. , Schilling William P.
Published: July 2002

Recent findings have suggested that the vertebrate trp family of channel proteins is the structural basis for Ca2+ influx through the capacitative calcium entry (CCE) pathway. We have discerned, in bovine aortic endothelial cells, the concomitant expression of four such vertebrate genes: trp‐1 (two splice variants), trp‐3, trp‐4 and trp‐5. Exogenous hormones rendered dynamic effects on the transcript levels of these genes. Most notably, β‐estradiol significantly down‐regulated trp‐4 while trans‐retinoic acid dramatically up‐regulated trp‐5; yet these hormones rendered little change in CCE. These findings suggest that the extent of a given trp channel's participation in CCE is not reflected in alterations of its transcript level.
Identification of Myo3, a second type‐II myosin heavy chain in the fission yeast Schizosaccharomyces pombe
Authors: Motegi Fumio , Nakano Kentaro , Kitayama Chikako , Yamamoto Masayuki , Mabuchi Issei
Published: July 2002

We cloned the myo3
+ gene of Schizosaccharomyces pombe which encodes a type‐II myosin heavy chain. myo3 null cells showed a defect in cytokinesis under certain conditions. Overproduction of Myo3 also showed a defect in cytokinesis. Double mutant analysis indicated that Myo3 genetically interacts with Cdc8 tropomyosin and actin. Myo3 may be implicated in cytokinesis and stabilization of F‐actin cables. Moreover, the function of Myo2 can be replaced by overexpressed Myo3. We observed a modest synthetic interaction between Myo2 and Myo3. Thus, Myo2 and Myo3 seem to cooperate in the formation of the F‐actin ring in S. pombe.
Oxygen dependence of flux control of cytochrome c oxidase – implications for mitochondrial diseases
Authors: Wiedemann Falk R , Kunz Wolfram S
Published: July 2002

The oxygen dependence of cytochrome c oxidase control on succinate oxidation was investigated in saponin‐permeabilized muscle fibers and isolated mitochondria from mouse quadriceps muscle applying metabolic control analysis. For this cyanide titrations of the oxygen consumption in the presence of succinate+rotenone were performed at different oxygen concentrations in the medium. While with isolated mitochondria high flux control coefficients were detected only at oxygen concentrations close to the K
M value of cytochrome c oxidase, with saponin‐permeabilized fibers a significant increase of cytochrome c oxidase flux control was already observed below 130 μM oxygen. The result is in line with the high oxygen sensitivity of maximal respiration of saponin‐permeabilized muscle fibers (P50=33 μM) caused most probably by oxygen diffusion gradients through the fiber lattice. The oxygen dependence of cytochrome c oxidase flux control in muscle fibers can explain the pathological phenotype of mild cytochrome c oxidase deficiencies in mitochondrial myopathies.
Alternative splicing and tissue specific expression of the 5′ truncated bCCE 1 variant bCCE 1Δ514
Authors: Freichel Marc , Wissenbach Ulrich , Philipp Stephan , Flockerzi Veit
Published: July 2002

In many non‐excitable as well as electrically excitable cells, depletion of intracellular Ca2+ stores after stimulation of G protein coupled receptors or receptor tyrosine kinases is followed by Ca2+ entry across the plasma membrane, a mechanism referred to as capacitative calcium entry (CCE) [Putney, J.W., Cell Calcium 11 (1990) 611–624; Fasolato, C. et al., Trends Pharmacol Sci. 15 (1994) 77–83]. Recently, we reported that bCCE 1, a homologue of the Drosophila protein trp, exhibits the characteristics of CCE channels [Philipp, S. et al., EMBO J. 15 (1996) 6166–6171]. In this study, we report the cloning of a 5′ truncated splice variant (bCCE 1Δ514) of the full‐length bCCE 1. The bCCE 1Δ514 cDNA encodes a protein of 486 amino acids with the ATG triplet encoding M514 of bCCE 1 as translation initiation codon and, therefore, comprises two putative transmembrane segments corresponding to the predicted transmembrane segments 5 and 6 of bCCE 1. bCCE 1Δ514 transcripts appear to be specifically expressed in the adrenal gland and genome analysis reveals an alternative splice site within an exon of the CCE 1 gene leading to the formation of bCCE 1Δ514.
The cytotoxin α‐sarcin behaves as a cyclizing ribonuclease
Authors: Lacadena Javier , Martı́nez del Pozo Alvaro , Lacadena Valle , Martı́nez-Ruiz Antonio , Mancheño José M , Oñaderra Mercedes , Gavilanes José G
Published: July 2002

The hydrolysis of adenylyl(3′→5′)adenosine (ApA) and guanylyl(3′→5′)adenosine (GpA) dinucleotides by the cytotoxic protein α‐sarcin has been studied. Quantitative analysis of the reaction has been performed through reverse‐phase chromatographic (HPLC) separation of the resulting products. The hydrolysis of the 3′‐5′ phosphodiester bond of these substrates yields the 2′‐3′ cyclic mononucleotide; this intermediate is converted into the corresponding 3′‐monophosphate derivative as the final product of the reaction. The values of the apparent Michaelis constant (K
M), k
cat and k
cat/K
M have also been calculated. The obtained results fit into a two‐step mechanism for the enzymatic activity of α‐sarcin and allow to consider this protein as a cyclizing RNase.
A somatostatin receptor 1 selective ligand inhibits Ca2+ currents in rat insulinoma 1046‐38 cells
Authors: Roosterman Dirk , Glassmeier Günter , Baumeister Hans , Scherübl Hans , Meyerhof Wolfgang
Published: July 2002

Rat insulinoma 1046‐38 cells represent a model system to study β‐cell function. The mRNAs for sst1 and sst2, two of the five somatostatin receptors, were detected by reverse transcription polymerase chain reaction amplification in these cells. Displacement binding analysis suggested that sst1 represents the major somatostatin receptor subtype. The sst1 selective compound CH‐275 did not inhibit adenylyl cyclases while compounds that activated sst2 did. In contrast, CH‐275 caused a marked inhibition of voltage‐operated Ca2+ channels while the sst2 specific analog octreotide elicited a less pronounced effect suggesting that in rat insulinoma 1046‐38 cells sst1 preferably mediates the inhibition of Ca2+ channels.
A cosmid and cDNA fine physical map of a human chromosome 13q14 region frequently lost in B‐cell chronic lymphocytic leukemia and identification of a new putative tumor suppressor gene, Leu5
Authors: Kapanadze Bagrat , Kashuba Vladimir , Baranova Anna , Rasool Omid , van Everdink Wim , Liu Yie , Syomov Alexander , Corcoran Martin , Poltaraus Andrey , Brodyansky Vadim , Syomova Natalia , Kazakov Alexey , Ibbotson Rachel , van den Berg Anke , Gizatullin Rinat , Fedorova Ludmila , Sulimova Galina , Zelenin Alexander , Deaven Larry , Lehrach Hans , Grander Dan , Buys Charles , Oscier David , Zabarovsky Eugene R. , Einhorn Stephan , Yankovsky Nick
Published: July 2002

B‐cell chronic lymphocytic leukemia (B‐CLL) is a human hematological neoplastic disease often associated with the loss of a chromosome 13 region between RB1 gene and locus D13S25. A new tumor suppressor gene (TSG) may be located in the region. A cosmid contig has been constructed between the loci D13S1168 (WI9598) and D13S25 (H2‐42), which corresponds to the minimal region shared by B‐CLL associated deletions. The contig includes more than 200 LANL and ICRF cosmid clones covering 620 kb. Three cDNAs likely corresponding to three different genes have been found in the minimally deleted region, sequenced and mapped against the contigged cosmids. cDNA clone 10k4 as well as a chimeric clone 13g3, codes for a zinc‐finger domain of the RING type and shares homology to some known genes involved in tumorigenesis (RET finger protein, BRCA1) and embryogenesis (MID1). We have termed the gene corresponding to 10k4/13g3 clones LEU5. This is the first gene with homology to known TSGs which has been found in the region of B‐CLL rearrangements.
Regulation of gelatinase B (MMP‐9) in leukocytes by plant lectins
Authors: Dubois Bénédicte , Peumans Willy J , Van Damme Els J.M , Van Damme Jo , Opdenakker Ghislain
Published: July 2002

The stimulatory or inhibitory effects of plant lectins on the production of gelatinase A (MMP‐2) and gelatinase B (MMP‐9) by mononuclear white blood cells was investigated by substrate zymography. Leukocyte cultures from 24‐h old buffy coats were spontaneously activated and produced high levels of gelatinase B. Using such cultures the suppressing activity of the Datura stramonium, Viscum album, Bauhinia purpurea, Triticum aestivum and Maackia amurensis lectins on gelatinase B induction were demonstrated. When fresh leukocyte preparations from single blood donors were used, low levels of gelatinase B were produced. The induction of gelatinase B was confirmed for concanavalin A and phytohaemagglutinin (PHA‐L4). In addition, the Urtica dioica, Calystegia sepium, Convolvulus arvensis and Colchicum autumnale lectins were documented as novel and potent inducers of gelatinase B. Since high circulating gelatinase B levels are associated with specific pathologies, including shock syndromes, the acute toxicity of many lectins might be partially mediated or influenced by gelatinase induction.
The carboxy‐terminal domain of the receptor‐associated protein binds to the Vps10p domain of sortilin
Authors: Tauris Jacob , Ellgaard Lars , Jacobsen Christian , Nielsen Morten S , Madsen Peder , Thøgersen Hans C , Gliemann Jørgen , Petersen Claus Munck , Moestrup Søren K
Published: July 2002

Binding of the receptor‐associated protein (RAP) to the newly identified putative sorting receptor, sortilin, was analyzed by surface plasmon resonance analysis of recombinant RAP and sortilin domains and compared with binding to megalin and low density lipoprotein receptor‐related protein (LRP). The data show that the RAP‐binding site in sortilin is localized in the cysteine‐rich lumenal part homologous to yeast vacuolar protein‐sorting 10 protein (Vps10p), and the sortilin‐binding site in RAP is localized in the carboxy‐terminal domain III of the three homologous domains in RAP. Whereas sortilin bound only RAP domain III, megalin and LRP bound all RAP domains with the functional affinity order: domain III>domain I>domain II.
Oxidative decomposition of malonic acid as basis for the action of manganese peroxidase in the absence of hydrogen peroxide
Authors: Hofrichter M. , Ziegenhagen D. , Vares T. , Friedrich M. , Jäger M.G. , Fritsche W. , Hatakka A.
Published: July 2002

Manganese peroxidase (MnP) from the ligninolytic basidiomycetesPhlebia radiataandNematoloma frowardiiwas found to decompose malonate oxidatively in the absence of H2O2in a reaction system consisting of the enzyme, sodium malonate and MnCl2. The enzymatic oxidation resulted in a substantial decrease in malonate concentration and the formation of CO2, oxalate, glyoxylate and formate. Simultaneously with the decomposition of malonate, Mn(II) was oxidized to Mn(III) leading to high transient concentrations of the latter. MnP action in the absence of H2O2started slowly after a lag period of 3 h. The lag period was considerably shortened after a single addition of Mn(III). Superoxide dismutase and catalase inhibited the enzymatic reaction partly, ascorbate completely. ESR studies demonstrated the formation of a carbon‐centered radical during the course of the reaction. We propose that the latter generates peroxides that can be used by MnP to oxidize Mn(II) to Mn(III).
Matrilin‐4, a new member of the matrilin family of extracellular matrix proteins1
Authors: Wagener Raimund , Kobbe Birgit , Paulsson Mats
Published: July 2002

Mouse cDNA encoding for matrilin‐4 was cloned and the primary structure of this fourth member of the matrilin family was deduced from the nucleotide sequence. The protein precursor of 624 amino acids consists of a putative signal peptide, two vWFA‐like domains linked by four epidermal growth factor‐like modules and a potential coiled‐coil α‐helical oligomerization domain at the C‐terminus. The predicted M
r of the mature protein is 66 442. Expression in lung, brain, sternum, kidney and heart was detected by Northern blot analysis of mouse mRNA. Additionally an alternatively spliced mRNA lacking the sequence coding for the first vWFA domain was found in 7 weeks old mice leading to a protein precursor of 434 amino acids and a predicted M
r of the mature protein of 45 468.
Identification of a possible MAP kinase cascade in Arabidopsis thaliana based on pairwise yeast two‐hybrid analysis and functional complementation tests of yeast mutants
Authors: Mizoguchi Tsuyoshi , Ichimura Kazuya , Irie Kenji , Morris Peter , Giraudat Jérôme , Matsumoto Kunihiro , Shinozaki Kazuo
Published: July 2002

A possible MAP kinase (MAPK) cascade of Arabidopsis thaliana was identified on the basis of both yeast 2‐hybrid analysis and complementation analysis of yeast mutants. Specific protein‐protein interactions between ATMPK4 (a MAPK) and MEK1 (a MAPKK) and interactions between MEK1 and ATMEKK1 (a MAPKKK) were detected by using the 2‐hybrid system. A growth defect of the yeast mpk1Δ mutant was reversed by coexpression of ATMPK4 and MEK1. Coexpression of the N‐terminal deletion form of ATMEKK1 increased the ability of MEK1 to suppress a growth defect of the yeast pbs2Δ mutant. These results suggest that ATMPK4, MEK1, and ATMEKK1 may interact with each other and constitute a specific MAPK cascade in Arabidopsis. This is the first demonstration of a possible MAPK cascade in plants.
Origin of superoxide production by endothelial nitric oxide synthase
Authors: Stroes E , Hijmering M , van Zandvoort M , Wever R , Rabelink T.J , van Faassen E.E
Published: July 2002

Using fluorescence optical and electron spin resonance spectroscopy, we have investigated the production of superoxide by bovine endothelial nitric oxide synthase (NOS). In contrast to neuronal NOS, the heme moiety is identified as the exclusive source of superoxide production by endothelial NOS. Thus, calmodulin‐mediated enzyme regulation affects production of nitric oxide and superoxide simultaneously and inseparably. The balance between the nitric oxide/superoxide reaction pathways may be shifted by addition of exogenous heme‐specific agents, such as tetrahydrobiopterin. Our results have direct relevance for the pathophysiology of atherosclerosis.
Effects of cold acclimation and palmitate on energy coupling in duckling skeletal muscle mitochondria
Authors: Roussel Damien , Rouanet Jean-Louis , Duchamp Claude , Barré Hervé
Published: July 2002

Gastrocnemius subsarcolemmal and intermyofibrillar mitochondria were isolated from 5‐week‐old cold‐acclimated and thermoneutral control ducklings. In vitro respiration (polarography) and ATP synthesis (bioluminescence) were determined at 25°C. Subsarcolemmal mitochondria showed a higher state 4 respiration and lower respiratory control and ADP/O ratio in cold‐acclimated than in thermoneutral ducklings. Palmitate decreased the rate of ATP synthesis in both mitochondrial populations to about 30% of maximal but failed to abolish this process even at high concentrations. It is suggested that both expensive ATP synthesis and increased ATP hydrolysis could contribute synergistically to muscle non‐shivering thermogenesis in cold‐acclimated ducklings.