bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular basis for different rates of recovery from inactivation in the Shaker potassium channel family
Authors: Wittka Reilinde , Stocker Martin , Boheim Günther , Pongs Olaf
Published: July 2002

The two alternative carboxyl‐termini of Shaker K+ channels strongly influence the rates of inactivation and of recovery from channel inactivation. We show that this distinct inactivation behaviour is due to an alanine/valine amino acid replacement within the Shaker carboxyl‐terminus at a site that occurs within the proposed membrane spanning segment S6.
ADPG formation by the ADP‐specific cleavage of sucrose‐reassessment of sucrose synthase
Authors: Pozueta-Romero Javier , Yamaguchi Junji , Akazawa Takashi
Published: July 2002

The standardized enzyme coupling method for assaying sucrose synthase activities in the direction of sucrose cleavage was reexamined using enzyme preparations from cultured cells of sycamore (Acer pseudoplatanus L.) and spinach leaves (Spinacea oleracea). Both ATP and Tris, commonly utilized in assay systems to measure sucrose synthase, were found to inhibit non‐competitively the ADPG‐synthesizing activities of the enzyme. Upon substituting ATP by either GTP or UTP, and Tris by HEPES, we found that the sucrose synthase is capable of producing ADPG effectively, recognizing ADP as the principal substrate (K
m = 5.3 μM (sycamore) and 16.8 μM (spinach)). The V
max value for the synthesis of ADPG clearly surpasses the V
max observed for the synthesis of UDPG by the enzyme, It was found that UDP is not inhibitory on the synthesis of ADPG by SS, which behaves allosterically with respect to the concentration level of sucrose.
Glutamic acid‐112 of the A subunit of heat‐labile enterotoxin from enterotoxigenic Escherichia coli is important for ADP‐ribosyltransferase activity
Authors: Tsuji Takao , Inoue Takashi , Miyama Akio , Noda Masatoshi
Published: July 2002

A mutant strain of enterotoxigenic Escherichia coli (E. coli pTUH 6A) produced an abnormal heat‐labile enterotoxin (LT), the A subunit of which has a single amino acid substitution at position 112 (Glu‐112 to Lys‐112). As already reported, this mutant LT had no ileal loop and vascular permeability activities [(1990) J. Biol. Chem. 265, 22520–22525]. In this paper we report that the mutant LT showed no CHO cell elongation activity and did not activate adenylate cyclase of target cells. Moreover, no ADP‐ribosyltransferase activity was detected in the mutant LT. It is concluded that the amino acid substitution at position 112 abolished the ADP‐ribosyltransferase activity of the A subunit and this leads to the loss of toxic activities of LT.
Evidence for non‐cysteinyl coordination of the [2Fe‐2S] cluster in Escherichia coli succinate dehydrogenase
Authors: Werth Mark T. , Sices Harry , Cecchini Gary , Schröder Imke , Lasage Susanne , Gunsalus Robert P. , Johnson Michael K.
Published: July 2002

The consequences of replacing Cys65 in the FrdB subunit of Escherichia coli fumarate reductase by Asp or Ala have been investigated in terms of bacterial growth, enzymatic activity, and the EPR/redox properties of the [2Fe‐2S] cluster. An aspartic acid residue occupies the equivalent position in E. coli succinate dehydrogenase, and the FrdBCys65Asp mutation has little effect on cell growth, enzyme activity or the physical properties of the Frd [2Fe‐2S] cluster. In contrast, the [2Fe‐2S] cluster was not observed in the FrdBCys65Ala mutant showing that a coordinating residue is required at this position for assembly of this cluster and significant levels or enzymatic activity. These results support the presence of one non‐cysteinyl, oxygenic ligand for the [2Fe‐2S] cluster in E. coli succinate dehydrogenase.
Amino acid sequence determination of Ancrod, the thrombin‐like α‐fibrinogenase from the venom of Akistrodon rhodostoma
Authors: Burkhart William , Smith Gardiner F.H. , Su Jui-Lan , Parikh Indu , LeVine Harry
Published: July 2002

The thrombin‐like serine protease and antithrombotic agent. Ancrod, was rapidly purified from the crude venom of Akistradon rhodostoma by agmatine‐Sepharose affinity chromatography followed by MonoQ anion exchange chromatography. N‐Terminal sequencing and analysis of overlapping proteolytic fragments of purified Ancrod by automated Edman degradation in combination with tandem mass spectroscopy allowed the determination of the 234 amino acid sequence of the protease. Glycosylation sites at all five canonical N‐linked glycosylation sites were inferred from the appearance of blank sequencer cycles in the amino acid sequence and were confirmed by mass spectroscopic analysis of the N‐glycanase‐treated peptides. Monoclonal antibodies raised against the denatured protein and HF‐deglycosylated protein recognized Ancrod on Western blots. Sequence comparison to other thrombin‐like serine proteases and reptilian fibrinogenases revealed a number of similarities, most notably the catalytic triad and many conserved cysteine positions.
Endothelin 1 hydrolysis by rat kidney membranes
Authors: Yamaguchi Toru , Fukase Masaaki , Arao Makoto , Sugimoto Toshitsugu , Chihara Kazuo
Published: July 2002

Hydrolysis of endothelin 1 by rat kidney membranes was investigated using a reverse‐phase HPLC and an automated gas‐phase protein sequencer. Endothelin 1 was hydrolyzed into four major fragments which were detected by HPLC, Phosphoramidon, an inhibitor of neutral endopeptidase 24,11, almost completely suppressed the production of three fragments, but one fragment was not affected by the inhibitor. Analysis of N‐terminal sequences of the degradation products revealed that the phosphoramidon‐sensitive fragments were generated by cleavage at the Ser5–Leu6 bond of endothelin 1 that was identical with its cleavage site by purified rat endopeptidase 24,11, reported previously. The phosphoramidon‐insensitive fragment was produced by cleavage at Leu17–Asp18, which was distinct from the sites by endopeptidase 24,11, but corresponded to that by a phosphoramidon‐insensitive metallo‐endopeptidase recently isolated from rat kidney membranes by μs [(1992) Eur. J. Biochem. 204, 547–552]. Kinetic determination of endothelin 1 hydrolysis by the isolated enzyme yielded values of Km
=71.5 μM and k
cal=1.49 s−1, giving a ratio of kcal K
m
=2.08 × 104 s−1·M−1. The Km
value was much higher and the k
cal/K
m
value was much lower than those for rat endopeptidase 24,11 reported previously. Thus, endopeptidase 24,11 appears to hydrolyze endothelin 1 more efficiently than the isolated enzyme does. Both enzymes may play physiological roles in the metabolism of endothelin 1 by rat kidney membranes in vivo.
The role of the C‐terminal region of rat brain natriuretic peptide in receptor selectivity
Authors: Shimekake Yoshiyuki , Kawabata Tomoji , Nakamura Masuhisa , Nagata Kiyoshi
Published: July 2002

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) have different C‐terminal tail structures compared with the rather conservative ring structures which consist of 17 amino acid residues. To examine the different effects of the tail structures of ANP and BNP on their interaction with receptors, we synthesized several peptide analogs and measured their biological actions in three different assay systems. Deletion of the C‐terminal tail from rat BNP did not effect the vasorelaxation activity against rat aorta, but it promoted cGMP production in cultured rat aortic smooth muscle cells (RASMC). Deletion of the C‐terminal tail from rat ANP diminished both vasorelaxant and cGMP producing activities. In a binding competition assay with RASMC and [125I]rat ANP‐(1–28), the competition activities of both ANP and BNP were greatly reduced by C‐terminal deletion. In addition, we obtained agonists with novel receptor selectivity.
ATP binding to bovine serum albumin
Authors: Bauer Michael , Baumann Joachim , Trommer Wolfgang E.
Published: July 2002

Specific binding or ATP to bovine serum albumin (BSA) is demonstrated employing ATP derivatives spin‐labeled at either N6 or C8 of adenine ring or at the ribose moiety. Based on a 1:1 stoichiometry binding constants are in the 50–100 μM range. Binding is largely competitive with ATP or stearic acid. A small fraction of the labeled nucleotides could not be liberated by these ligands. Binding of AMP is in the millimolar range, only.
Overexpression of protein kinase C‐ϵ enhances the stimulatory effect of ethanol on phospholipase C‐mediated hydrolysis of phosphatidylethanolamine in NIH 3T3 fibroblasts
Authors: Kiss Zoltan , Garamszegi Nandor
Published: July 2002

Previously, ethanol and the protein kinase C (PKC) activators phorbol 12‐myristate 13‐acetate (PMA) and bombesin were shown to synergistically stimulate phospholipase C (PLC)‐mediated hydrolysis of phosphatidylethanolamine (PtdEtn) in NIH 3T3 fibroblasts. Here we used fibroblasts overexpressing PKC‐ϵ 15‐fold to examine the possible role of this enzyme in the regulation of PtdEtn hydrolysis by ethanol. Overexpressed PKC‐ϵ (i) greatly enhanced the stimulatory effects of ethanol (37.5–150 mM) on PLC‐mediated PtdEtn hydrolysis, and (ii) eliminated the need for the co‐presence of a PKC activator for maximal (3,3‐fold) stimulation of PLC by 150 mM ethanol. Results suggest that PKC‐ϵ is a potential positive regulator of the PtdEtn‐hydrolyzing PLC activity, and that the functional interaction between PKC‐ϵ and PLC is facilitated by ethanol.
Activation of NF‐κB by cAMP in human myeloid cells
Authors: Serkkola Elina , Hurme Mikko
Published: July 2002

Nuclear factor kappa B (NF‐κB) is a DNA‐binding regulatory factor that controls the transcription of a number of genes. Various agents are known to activate this factor. We have studied the ability of cAMP to stimulate NF‐κB activity in human myeloid cells. Electrophoretic mobility assay revealed that structural cAMP analogs and agents elevating intracellular cAMP levels induced NF‐κB DNA‐binding activity. The inducibility was dependent on the maturation stage of myeloid cells. In promyelocytic HL‐60 cells cAMP induced higher NF‐κB activity than in more differential THP‐1 cells and in human monocytes. By transfecting HL‐60 and THP‐1 cells with reporter constructs containing NF‐κB DNA‐binding sites, we observed that cAMP‐induced NF‐κB was transcriptionally active.
Specificity determinants for the AMP‐activated protein kinase and its plant homologue analysed using synthetic peptides
Authors: Weekes John , Ball Kathryn L. , Caudwell F.Barry , Hardie D.Grahame
Published: July 2002

Inspection of sequences around sites phosphorylated by the AMP‐activated protein kinase (AMP‐PK), and homologous sequences from other species, indicates conserved features. There are hydrophobic residues (M, V, L, I) at P‐5 and P+4, and at least one basic residue (R, K, H) at P‐2, P‐3 or P‐4. The importance of these residues has been established for AMP‐PK and its putative plant homologue using a series of synthetic peptides. These results confirm the functional similarity of the animal and plant kinases, and suggest that the required motif for recognition of substrate by either kinase is M/V/L/I‐(R/K/H,X,X)‐X‐S/T‐X‐X‐X‐M/V/L/I.
Hydrolysis of the IciA protein, an inhibitor of DNA replication initiation, by protease Do in Escherichia coli
Authors: Yoo Soon Ji , Seol Jae Hong , Woo Seung Kyoon , Suh Se Won , Hwang Deog Su , Ha Doo Bong , Chung Chin Ha
Published: July 2002

The 33 kDa IciA protein, an inhibitor of replication initiation of the Escherichia coli chromosome, was found to be specifically cleaved to 27 kDa fragment by protease Do, the htrA gene product. The 27 kDa polypeptide could no longer interact with the oriC region, and therefore the cleavage‐site is likely to reside within the N‐terminal DNA‐binding domain of the IciA protein. In addition, protease Do was found to localize primarily to the cytoplasm although it also could bind to membranes through an ionic interaction. These results suggest that intracellular breakdown of the IciA protein by protease Do may provide a potential mechanism involving the regulation of initiation of DNA replication in Escherichia coli.
Modulation of mammalian cell proliferation by a modified tRNA base of bacterial origin
Authors: Langgut Werner , Reisser Thomas , Nishimura Susumu , Kersten Helga
Published: July 2002

Addition of the q‐base to q‐deflcient non‐transformed mammalian cells stimulated their proliferation. The q‐base also improved proliferation of some cancer‐derived cell lines, but inhibited proliferation of others. The proliferation of HeLa‐S3 carcinoma cells was stimulated by q under aerobic conditions, but was inhibited when the cells had shifted their energy metabolism towards glycolysis as the result of oxygen limitation. Q‐deficient cells could not adapt their proliferation to the respective oxygen tension. The q‐base stimulated the proliferation of non‐transformed fibroblasts but inhibited proliferation of the same cell line, when aerobic glycolysis was increased after transformation with the ras gene. The results suggest that the q‐base permits mammalian cells to adapt their proliferation to their specific metabolic state.
Antisense c‐myc oligonucleotide promotes chondrogenesis and enhances RA responsiveness of mouse limb mesenchymal cells in vitro
Authors: Motoyama Jun , Eto Kazuhiro
Published: July 2002

To examine the role of c‐myc protein during chondrogenesis, we exposed 11 day p.c. mouse limb mesenchymal cells to the antisense c‐myc oligonucleotide in micromass culture. The antisense oligonucleotide inhibited the c‐myc protein expression, and intensely promoted chondrogenesis in the exposed cells. Most of the cells differentiated into cartilaginous cells, whereas they differentiated into cartilaginous and fibrous cells under the control conditions. The antisense oligonucleotide increased the inhibitory efficiency of all‐trans retinoic acid (RA) to the chondrogenesis. These results suggest that the c‐myc protein suppress the chondrogenesis and reduces RA responsiveness in the limb mesenchymal cells.
Different induction mechanisms of mRNA for inducible nitric oxide synthase in rat smooth muscle cells in culture and in aortic strips
Authors: Sirsjo Allan , Söderkvist Peter , Sundqvist Tommy , Carlsson Maria , Öst Martin , Gidlöf Anders
Published: July 2002

The expression of mRNA for the inducible form of nitric oxide synthase, (iNOS), was studied in rat aortic smooth muscle cells, (SMCs) in cell culture and in strips of rat aorta by reverse transcriptase coupled to the polymerase chain reaction. iNOS mRNA expression was weak in cultured SMCs when exposed to either interferon‐γ (IFNγ) or lipopolysaccharide (LPS), but the combination LPS + IFNγ enhanced the expression. In aortic strips LPS alone induced a pronounced expression, with no further increase by IFNγ. Cycloheximide potentiated the expression of iNOS mRNA in SMCs in culture stimulated with LPS + IFNγ but attenuated the response in aortic strips.The results indicate different cellular signaling pathways for the induction of iNOS mRNA by LPS and/or IFNγ, in cultured SMCs and in rat aortic strips.
Cloning and expression of the bovine sodium‐ and chloride‐dependent noradrenaline transporter
Authors: Lingen Bettina , Brüss Michael , Bönisch Heinz
Published: July 2002

A cDNA encoding a functional bovine, tricyclic antidepressant‐sensitive noradrenaline transporter has been identified by screening a λgt11 cDNA library of the bovine adrenal medulla using the cDNA of the human noradrenaline transporter [1991, Nature 350, 350‐354]. The sequence predicts a protein of 615 amino acids (M
r, 68 900). The bovine transporter shares 93% amino acid identity with the human sequence, but displays two more consensus sites for phosphorylation by protein kinase C. Transient expression of the transporter in COS‐7 cells resulted in a sodium‐ and chloride‐dependent uptake of noradrenaline with a pharmacology typical for a neuronal noradrenaline transporter.
History and epidemiology of panic
Authors: Angst J
Published: July 2002

The conceptualisation and investigation of panic disorder have developed on two different axes, medical and psychological, for more than 100 years. In medicine, reports of acute anxiety attacks with cardiac, circulatory, and respiratory symptoms date back to the French Revolution, and terms such as “soldier's heart”, “neurocirculatory neurasthenia”, or “hyperventilation syndrome” have been used to describe them. In psychological medicine, anxiety attacks were first reported by Domrich in 1849. These attacks, which were thought to be caused by strong emotions, were classified mainly within neurasthenia, until Freud created the concept of anxiety neurosis in 1895.
Epigenes: design and construction of new hereditary units
Authors: Tchuraev R.N , Stupak I.V , Tropynina T.S , Stupak E.E
Published: July 2002

A plasmid digene construction designed before [Tchuraev, R.N. (1982) J. Gen. Biol. 43, 79–87] has been realised, including feedback by repressing proteins with given trigger regime of gene functioning. Experimental tests of the expected epigene properties of the obtained pECPI recombinant plasmid involving lacI and cI857
regulatory genes have shown a phenomenon of steady inheritance of two alternative epigenotypes lacI1cI0
and lacI0cI1
, as well as an external toggle switch through metabolitic and temperature signals from one inherited functional state of the cyclic digene system into another. Thus, we have constructed a hereditary unit of a specific kind, namely, a two‐component stationary epigene with preset properties.
Functional redundancy of the zinc fingers of A20 for inhibition of NF‐κB activation and protein–protein interactions
Authors: Klinkenberg Marco , Van Huffel Sofie , Heyninck Karen , Beyaert Rudi
Published: July 2002

The tumor necrosis factor (TNF) inducible protein A20 is a potent inhibitor of nuclear factor‐κB (IκB)‐mediated gene expression in response to TNF and several other stimuli. The C‐terminal domain of A20 is characterized by seven zinc finger structures. Here, we show that a minimum of four zinc fingers is required to inhibit TNF‐induced nuclear factor‐κB (NF‐κB) activation to a level that is comparable to that obtained with the wild‐type A20 protein. However, there was no strict requirement for a particular zinc finger structure, since a mutant A20 protein containing only the first four zinc fingers was as potent as a mutant protein containing only the last four zinc fingers. A similar functional redundancy of the A20 zinc fingers was also observed for binding of A20 to a number of other proteins, including two novel NF‐κB inhibitory proteins (ABIN‐1, ABIN‐2), A20 itself, the anti‐apoptotic protein TXBP151, and a regulatory component of the IκB kinase complex, IKKγ. Moreover, we demonstrate that complete loss of binding of any of these proteins correlates with complete loss of A20's ability to inhibit TNF‐induced NF‐κB activation. However, binding of IKKγ as such is not sufficient for inhibition of NF‐κB dependent gene expression in response to TNF.
Casein kinase II phosphorylates Ser468 in the PEST domain of the Drosophila IκB homologue cactus
Authors: Packman Leonard C , Kubota Ken , Parker James , Gay Nicholas J
Published: July 2002

Cactus protein is a Drosophila homologue of the mammalian IκB family of cytoplasmic anchor proteins. In unstimulated cells they function to retain rel/NFκB transcription factors in the cytoplasm but are rapidly degraded in response to signalling. The destruction of cactus or IκBα allows the rel/NFκB transcription factor to relocalise to the nucleus. Cactus is a phosphoprotein and has in its C‐terminus a PEST protein stability domain. In this paper we show that, like mammalian IκBα, the PEST domain of cactus is phosphorylated by casein kinase II. We have localised the site of modification to a single residue, Ser468, and find no evidence for additional phosphorylation sites. The conservation of these sites in mammalian and invertebrate cytoplasmic anchor proteins suggests that phosphorylation by casein kinase II may play a critical functional role, plausibly in the regulation of constitutive or inducible proteolysis.
Secondary structure of the IIB domain of the Escherichia coli mannose transporter, a new fold in the class of α/β twisted open‐sheet structures
Authors: Gschwind Ruth M. , Gemmecker Gerd , Leutner Michael , Kessler Horst , Gutknecht Regula , Lanz Regina , Flükiger Karin , Erni Bernhard
Published: July 2002

© 1997 Federation of European Biochemical Societies.
cDNA cloning and expression of pokeweed antiviral protein from seeds in Escherichia coli and its inhibition of protein synthesis in vitro
Authors: Poyet Jean-Luc , Hoeveler Arnd
Published: July 2002

Pokeweed antiviral proteins (PAP) represent a family of protein toxins isolated from various organs and at different stages of development of Phytolacca americana (pokeweed). We isolated, sequenced and characterized for the first time a complete cDNA encoding a pokeweed antiviral protein expressed in seeds. The cDNA of PAP‐S consists of 1249 nucleotides and encodes a mature 262 amino acid protein. Its predicted amino acid sequence is more similar to PAP (76%) than to PAP II (31%). It is known from literature that PAP‐S is more active in inhibiting protein synthesis than other members of the PAP family. Therefore, the cDNA of PAP‐S was expressed in Escherichia coli and the biological activity of the recombinant protein was compared with that of PAP purified from spring leaves. In a rabbit translation system, the median inhibitory concentrations (IC50) of recombinant PAP‐S and native PAP were determined as 0.07 and 0.29 nM, respectively. Although the PAP‐S protein in seeds is glycosylated, PAP‐S can be expressed in Escherichia coli in a very active form, indicating that post‐translational modification in pokeweed does not seem to alter its ability to inhibit protein synthesis.
An improved purification of ECF1 and ECF1F0 by using a cytochrome bo‐deficient strain of Escherichia coli facilitates crystallization of these complexes
Authors: Grüber Gerhard , Hausrath Andrew , Sagermann Martin , Capaldi Roderick A
Published: July 2002

A novel strategy, which employs a cytochrome bo‐lacking strain (GO104) and a modified isolation procedure provides an effective approach for obtaining much purer preparations of ECF1F0 than described previously, as well as for isolating homogeneous and protein‐chemically pure ECF1. ECF1 obtained in this way could be crystallized by vapor‐diffusion using polyethylene glycol (PEG) as a precipitant in a form suitable for X‐ray diffraction analysis. The crystals belong to the orthorhombic space group P212121, with lattice parameters a=110, b=134, and c=269 Å, and diffract to a resolution of at least 6.4 Å.
High‐affinity binding of the yeastcis‐Golgi t‐SNARE, Sed5p, to wild‐type and mutant Sly1p, a modulator of transport vesicle docking
Authors: Grabowski Reiner , Gallwitz Dieter
Published: July 2002

Docking of ER‐derived vesicles to thecis‐Golgi compartment in yeast requires vesicle and target membrane receptors (v‐SNAREs and t‐SNAREs) and the GTPase Ypt1p. The t‐SNARE Sed5p is complexed with Sly1p in vivo. The mutant form Sly1‐20p rescues Ypt1p‐lacking cells from lethality, suggesting an inhibitory function of Sly1p in v‐SNARE/t‐SNARE interaction. Using surface plasmon resonance spectroscopy, we found that Sed5p binds Sly1p and Sly1‐20p with equally high affinity (KD=5.13×10−9M and 4.74×10−9M, respectively). Deletion studies show that the N‐terminal half of Sly1p rather than the C‐terminus (harbouring the E532K substitution in Sly1‐20p) is most critical for its binding to Sed5p. These data appear to argue for an active rather than an inhibitory role of Sly1p in vesicle docking.
Involvement of u‐PA in the anti‐apoptotic activity of TGFβ for vascular smooth muscle cells
Authors: Herbert J.M , Carmeliet P
Published: July 2002

Previous studies suggest a role for the plasminogen or fibrinolytic system in the activation of latent‐transforming growth β (L‐TGFβ) into active TGFβ. In the present study, the anti‐apoptotic activity of TGFβ on cultured vascular smooth muscle cells (SMC) isolated from the aorta of transgenic mice with single inactivation of genes encoding the tissue‐type plasminogen activator (t‐PA−/−), urokinase‐type plasminogen activator (u‐PA−/−), urokinase receptor (u‐PAR−/−) or plasminogen (Plg−/−) genes was examined. Latent‐TGFβ inhibited serum deprivation‐induced apoptosis of SMC isolated from wild‐type and t‐PA−/− mice but failed to reduce apoptosis of SMC isolated from u‐PA−/−, u‐PAR−/− or Plg−/− mice. Active TGFβ, however, was able to inhibit serum deprivation‐induced apoptosis of these 5 cell types, indicating that u‐PA and/or plasmin were involved in the activation of L‐TGFβ. The anti‐apoptotic effect of L‐TGFβ could not be evoked by addition of exogenous t‐PA to u‐PA−/− cells, but was revealed by addition of exogenous u‐PA or plasmin. This effect was dependent on the catalytic activity of plasmin as revealed by the dose‐dependent inhibition of aprotinin or epsilon aminocaproic acid (EACA). These results therefore indicate that, at least in vitro, u‐PA‐mediated plasmin, through the generation of active TGFβ from L‐TGFβ, is required for the anti‐apoptotic activity of TGFβ on SMC.
Structural requirement of highly‐conserved residues in globins
Authors: Ota Motonori , Isogai Yasuhiro , Nishikawa Ken
Published: July 2002

Globins have remarkable sequence diversity, and yet maintain a common fold. In spite of the diversity, there are highly‐conserved residues at several sites. The conserved residues were examined in terms of the structural stability, by employing the pseudo‐energy functions of the structure/sequence compatibility method. The fitness of each residue type to the structural environment was evaluated at seven highly‐conserved sites: the Leu (at the B10 site), Phe (CD1), and Leu (F4) residues were found to fit their respective sites due to hydrophobic interactions; Pro (C2) stabilizes the N‐terminal edge of an α‐helical structure; and Phe (CD4) is stabilized by backbone hydrogen‐bonding to Phe (CD1). On the other hand, the other two residues, His (E7) and His (F8), are poorly suited to the sites from a structural viewpoint, suggesting that their conservation clearly results from a heme‐related functional requirement. The invariant Phe residue (CD1) has been suggested to be important for supporting the heme. The present analysis revealed that this residue is also well suited to the site in terms of energy.
Binding of multiple ligands to pleckstrin homology domain regulates membrane translocation and enzyme activity of β‐adrenergic receptor kinase
Authors: Touhara Kazushige
Published: July 2002

Pleckstrin homology (PH) domains are discrete structural modules present in numerous proteins involved in signal transduction processes. In the case of the β‐adrenergic receptor kinase (βARK), PH domain‐mediated binding of two ligands, the βγ subunits of heterotrimeric G proteins (Gβγ) and phosphatidylinositol 4,5‐bisphosphate (PIP2), has been shown to be required for the kinase function. In this study, the ability of Gβγ and PIP2 to affect membrane localization of βARK is used to define the ligand binding characteristics of the βARK PH domain. The binding of these ligands to the PH domain of the intact kinase is shown to be cooperative, Gβγ increasing the affinity of the PH domain for PIP2. Notably, although PIP2‐dependent membrane association of βARK is observed at high concentrations of this lipid, in the absence of Gβγ, no receptor phosphorylation is observed. Peptides derived from the receptor intracellular loop inhibit the receptor phosphorylation without affecting the membrane translocation of the kinase complex, suggesting that βARK activity does not necessarily correlate with the amount of βARK associated with the membrane. These results point to a distinct role for each PH domain ligand in βARK‐mediated receptor phosphorylation. Strikingly, the ligand binding characteristics of the isolated βARK PH domain fused to glutathione S‐transferase are very different from those of the PH domain of the intact kinase. Thus, in contrast to the native protein, the isolated PH domain binds Gβγ and PIP2 independently and with no apparent cooperativity. That protein environment plays an important role in determining the ligand binding characteristics of a particular PH domain highlights the potential risks of inferring mechanisms from studies of isolated PH domains.
Ectopic expression of lunatic Fringe leads to downregulation of Serrate‐1 in the developing chick neural tube; analysis using in ovo electroporation transfection technique
Authors: Sakamoto Kei , Nakamura Harukazu , Takagi Minoru , Takeda Shin'ichi , Katsube Ken-ichi
Published: July 2002

Lunatic Fringe (l‐Fng) is one of the vertebrate homologues of Drosophila Fringe, which interacts with the Notch signal pathway and regulates activation of the Notch ligands, Delta and Serrate. To elucidate the roles of l‐Fng in vertebrate neurogenesis, we transfected chick l‐Fng (C‐l‐Fng) to chick neural tube using the in ovo electroporation technique and examined the subsequent changes in expression of Notch‐related genes. We observed downregulation of C‐Serrate‐1 by ectopic C‐l‐Fng expression which implied that C‐l‐Fng acts on the vertebrate Notch pathway to regulate the expression of its ligand.
Purification of a novel endothelin‐converting enzyme specific for big endothelin‐3
Authors: Hasegawa Hiroshi , Hiki Kazuaki , Sawamura Tatsuya , Aoyama Takuma , Okamoto Yasuo , Miwa Soichi , Shimohama Shun , Kimura Jun , Masaki Tomoh
Published: July 2002

Endothelin‐3 (ET‐3), a potent vasoactive peptide, is considered to be produced from big ET‐3 by endothelin‐converting enzyme (ECE) like the other members of the endothelin family (ET‐1 and ET‐2). We purified a novel ECE from bovine iris microsomes. The purified enzyme, a 140 kDa protein by SDS‐PAGE analysis, converted big ET‐3 to ET‐3 but not big ET‐1, with a K
m value of 0.14 μM for big ET‐3. The conversion to ET‐3 was confirmed with sandwich EIA by monoclonal antibodies, the elution profile of HPLC, and intracellular calcium mobilization in CHO‐K1 cells expressing recombinant human ETB receptors. The conversion activity was inhibited by an inhibitor of neutral endopeptidase 24.11 (NEP) phosphoramidon. These results show that ECE‐3 purified from bovine iris is a novel metalloprotease totally different from ECE‐1 or ECE‐2, in that the enzyme is highly specific for big ET‐3.
Nuclear association of tyrosine‐phosphorylated Vav to phospholipase C‐γ1 and phosphoinositide 3‐kinase during granulocytic differentiation of HL‐60 cells
Authors: Bertagnolo Valeria , Marchisio Marco , Volinia Stefano , Caramelli Elisabetta , Capitani Silvano
Published: July 2002

The granulocytic differentiation of HL‐60 cells induced by all‐trans retinoic acid was accompanied by a progressive tyrosine phosphorylation of specific proteins in either cells or isolated nuclei. Among these phosphoproteins, we identified the Vav adaptor in whole cells as well as in the inner nuclear compartment, where the increase in its tyrosine phosphorylation level was more conspicuous. We also demonstrated the differentiation‐dependent association of nuclear phosphorylated Vav to phospholipase C‐γ1 and to the p85 regulatory subunit of phosphoinositide 3‐kinase. The role of the Vav/phospholipase C‐γ1/phosphoinositide 3‐kinase phosphoprotein complexes in the nuclei of HL‐60 induced to differentiate along the granulocytic lineage is discussed.
Visualization of caveolin‐1, a caveolar marker protein, in living cells using green fluorescent protein (GFP) chimeras
Authors: Volontè Daniela , Galbiati Ferruccio , Lisanti Michael P.
Published: July 2002

Caveolin‐1, a suspected tumor suppressor, is a principal protein component of caveolae in vivo. Recently, we have shown that NIH 3T3 cells harboring anti‐sense caveolin‐1 exhibit a loss of contact inhibition and anchorage‐independent growth. These observations may be related to the ability of caveolin‐1 expression to positively regulate contact inhibition. In order to understand the postulated role of caveolin‐1 in contact inhibition, it will be necessary to follow the distribution of caveolins in living cells in response to a variety of stimuli, such as cell density. Here, we visualize the distribution of caveolin‐1 in living normal NIH 3T3 cells by creating GFP‐fusion proteins. In many respects, the behavior of these GFP‐caveolin‐1 fusion proteins is indistinguishable from endogenous caveolin‐1. These GFP‐caveolin‐1 fusion proteins co‐fractionated with endogenous caveolin‐1 using an established protocol that separates caveolae‐derived membranes from the bulk of cellular membranes and cytosolic proteins, and co‐localized with endogenous caveolin‐2 in vivo as seen by immunofluorescence microscopy. We show here that as NIH 3T3 cells become confluent, the distribution of GFP‐caveolin‐1 and endogenous caveolin‐1 shifts to areas of cell‐cell contact, coincident with contact inhibition. However, unlike endogenous caveolin‐1, the levels of GFP‐caveolin‐1 expression are unaffected by changes in cell density, serum starvation, or growth factor stimulation. These results are consistent with the idea that the levels of endogenous caveolin‐1 are modulated by either transcriptional or translational control, and that this modulation is separable from density‐dependent regulation of the distribution of caveolin‐1. These studies provide a new living‐model system for elucidating the dynamic mechanisms underlying the density‐dependent regulation of the distribution of caveolin‐1 and how this relates to contact inhibition.
Existence of uncoupling protein‐2 antigen in isolated mitochondria from various tissues
Authors: Ježek Petr , Žáčková Markéta , Řeháková Zuzana , Růžička Michal , Borecký Jiří , Škobisová Eva , Brucknerová Jana , Garlid Keith D. , Gimeno Ruth E. , Tartaglia Louis A.
Published: July 2002

Antibodies against Escherichia coli‐expressed uncoupling protein‐2 (UCP2) and uncoupling protein‐3 (UCP3) were raised by operating the blotted proteins into the spleen of minipigs. The antisera reacted more intensively with the recombinant UCP2 and UCP3 than with uncoupling protein‐1 (UCP1) isolated from brown adipose tissue. Moreover, anti‐UCP2 and cross‐reacting anti‐UCP3 antibodies identified the presence of the UCP2/3 antigen in isolated mitochondria from rat heart, rat kidney, rat brain, rabbit epididymal white adipose tissue, hamster brown adipose tissue, and rabbit skeletal muscle. It has been concluded that UCP2 is expressed in these tissues (UCP3 in skeletal muscle); however their existence in mitochondria had not previously been demonstrated.
Accumulation of mature mRNA in the nuclear fraction of mammalian cells
Authors: Gondran Pierre , Amiot Franck , Weil Dominique , Dautry François
Published: July 2002

Little is known about the nuclear mRNA content of mammalian cells. In this study, we analyzed by Northern blotting with a panel of probes the nuclear and cytoplasmic fractions derived from several rodent cell lines. For most of the genes under study, mature mRNAs could easily be detected in the nuclear fraction and accumulated to higher levels than the corresponding precursors. In addition, significant differences in the nucleo‐cytoplasmic partition of mature mRNAs were observed between genes as well as between cell types (NIH 3T3, CTLL‐2, D3‐ES, PC‐12), indicating that this nuclear accumulation of mRNA is regulated. Thus, while it is usually considered that splicing is the limiting step of pre‐mRNA processing, these results point towards transport or nuclear retention of mRNA as a key determinant of nuclear mRNA metabolism.
Molecular cloning of EDG‐3 and N‐Shc genes from the puffer fish, Fugu rubripes, and conservation of synteny with the human genome
Authors: Yamaguchi Fuminori , Yamaguchi Kumiko , Tokuda Masaaki , Brenner Sydney
Published: July 2002

EDG‐3 is a receptor for sphingosine‐1‐phosphate mapped on human chromosome 9q22.1–q22.2. We used the compact Fugu genome for its linkage analysis. The Fugu EDG‐3 was composed of one intron and two exons, encoding a 384 amino acid protein that has 56.9% homology with the human EDG‐3. Approximately 3 kb apart, a neuronal Shc (N‐Shc) gene was identified. It spans 7 kb containing 12 coding exons, and has an overall 53.4% similarity with the human protein. We mapped the human N‐Shc gene to chromosome 9q21.3–q22.2. This is the first report of the genomic structure and the linkage of these two genes conserved between Fugu and human.
Release of a product of growth arrest‐specific gene 6 from rat platelets
Authors: Ishimoto Yoshikazu , Nakano Toru
Published: July 2002

A product of growth arrest‐specific gene 6 (Gas6) is known to be synthesized by growth‐arrested cells. In this study, we found that several rat tissues including platelets contain Gas6 and activation of the platelets with thrombin provoked the release of Gas6. ADP and collagen, which as well as thrombin stimulated release of ATP from platelets, also enhanced the release of Gas6, suggesting that the mechanism of its release was similar to that of ATP release. This study provides the first evidence of growth arrest‐independent secretion of Gas6 and suggests the involvement of Gas6 in vascular diseases as well as hemostasis.
An expandable anchor for fixation in osteoporotic bone
Authors: McKoy Brodie E. , An Yuehuei H.
Published: July 2002

With the aging population, osteoporosis and osteoporotic fractures are becoming more prevalent. Adequate screw fixation in this type of bone is difficult. Screws are often cemented in bone to help obtain purchase. However, cement augmentation is not without problems. Here we present an expandable anchor for fixation in osteoporotic bone that does not require cement. We compared the ultimate holding power of this expandable anchor to a standard solid screw of similar dimensions. The solid screw and the expandable anchor were both placed into fresh frozen lumbar vertebral bodies and pulled out using a mechanical testing system. The expandable anchor had a 74% greater holding power compared to the standard screw (P = 0.02). The expandable anchor provides a significant increase in holding power without the problems associated with cement. This prototype anchor is promising for fixation in osteoporotic bone and warrants further evaluation. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Expression of calbindin‐D28k and parvalbumin during development of rat's basolateral amygdaloid complex
Authors: Berdel Bożena , Moryś Janusz
Published: July 2002

Parvalbumin and calbindin‐D28k are calcium‐binding proteins, which are considered to be markers for certain populations of GABAergic neurons. Their correct development in the basolateral amygdaloid complex is critical for the proper emotional functioning in adult live of human and animals. Therefore, in this paper we describe the pattern of the morphological differentiation and distribution of immunoreactive elements of the parvalbumin and calbindin‐D28k in this complex on the basis of immunohistochemically stained material obtained from embryonic (E20) and postnatal (P0–P90) rat brains.

Calbindin‐D28k appeared early in the development, already in the prenatal life. At this time immunopositive reaction was visible only in cell bodies. However, during development the population of immunopositive neurons was divided into four types: (1) polygonal; (2) piriform‐like; (3) bipolar; and (4) pyramidal‐like. Two weeks after birth calbindin‐D28k immunoreactivity also appeared in neuropil. First, there were visible calbindin‐D28k positive fibers and granules that encircled unstained cell bodies and formed basket‐like structures. Subsequently, these granules appeared along proximal parts of unstained dendrites forming, so called ‘cartridges’. The distribution of calbindin‐D28k positive cells during postnatal life was rather homogenous throughout whole basolateral complex. Intensity of calbindin‐D28k immunoreactivity reached mature level on the 21st day after birth.

The maturation pattern of parvalbumin immunopositive elements followed the same sequence as calbindin‐D28k, but it started much later — since the 17th day after birth and reached mature appearance on the 30th day of life. Contrary to calbindin‐D28k, parvalbumin was not homogeneously distributed in the basolateral complex. Originally, parvalbumin was restricted to the magnocellular part of basolateral nucleus but it was finally expressed also in the parvicellular part of basolateral nucleus and the dorsolateral part of lateral nucleus.

The differences in development of these two calcium‐binding proteins indicate that parvalbumin and calbindin‐D28k play diverse roles during development and maturation of the basolateral amygdala.
Particular aspects of adverse event assessment in post marketing surveillance
Authors: Baier D
Published: July 2002

The main feature of observational studies is the representation of naturalistic treatment conditions. In contrast to clinical trials, they allow the evaluation and quantification of adverse event profiles of drugs under “real life” conditions. The price for this unquestionable chance is the proneness to distorting factors, which may aggravate the interpretation of the study results. Analysis of observational study results therefore has to control for potentially influential factors and reconsider possible alternatives explaining observed associations. The most important distorting factors, which should be taken into account during analysis and interpretation are under-reporting, event selection, bias, confounding and misusage? Authors and readers of such study results should be aware of this possible sources of error, in order to derive optimal benefit from this study approach.
Plasma viscosity and fibrinogen in relation to haemodynamic findings in chronic congestive heart failure
Authors: Hoffmeister Albrecht , Hetzel Jürgen , Sander Silvia , Kron Martina , Hombach Vinzenz , Koenig Wolfgang
Published: July 2002

The aim of this study was to investigate an inflammatory response indicated by fibrinogen and plasma viscosity in relation to haemodynamic and clinical findings of patients with stable CHF due to coronary heart disease (CHD).
Nickel‐Catalyzed Cross‐Coupling of 2‐Methoxynaphthalene with Methyl 4‐(5,5‐Dimethyl‐1,3,2‐Dioxaborinan‐2‐yl)Benzoate
Authors: Kawashima Yuki , Furukawa Takayuki , Chatani* Naoto , Tobisu* Mamoru
Published: April 2020

This chapter article the procedure for nickel‐catalyzed cross‐coupling of 2‐methoxynaphthalene with methyl 4‐(5,5‐dimethyl‐1,3,2‐dioxaborinan‐2‐yl)benzoate. It presents some of the important points to be considered, the conditions that need to be maintained, characterization data, and the reagents required, as well as the techniques used and the equipment setup that are vital to carrying out the process. The Suzuki‐Miyaura coupling of aryl halides and their equivalents with arylboronic acids is recognized as one of the most powerful methods for forming aryl‐aryl bonds, which are pervasive in a number of fields of organic chemistry ranging from pharmaceuticals to advanced organic materials. The cross‐coupling of phenols can be classically achieved by their conversion to aryl triflates, thereby activating the C(aryl)‐O bond toward oxidative addition to a metal catalyst. The article also describes the hazards associated with working with chemicals and the ways to deal with these hazards.
Discussion Addendum For: The Direct Acyl‐Alkylation of Arynes. Preparation of Methyl 2‐(2‐acetylphenyl)acetate
Authors: Wright Austin C. , Stoltz* Brian M.
Published: April 2020

The direct insertion of aryne and heteroaryne moieties into carbon–carbon and carbon–heteroatom σ bonds presents an intriguing and unique strategy for the rapid functionalization of aryl and heteroaryl systems. This discussion addendum is intended to document advances made in the field of (hetero)arynes. Topics covered are recent methods for the acyl‐functionalization of arynes, syntheses of related strained systems, applications in heterocycle synthesis, miscellaneous transformations, and strategic uses in natural product synthesis. The addendum presents a supplemental review on aryne insertions into σ bonds. There has been one report harnessing arynes and cyclic alkynes for the asymmetric construction of all‐carbon quaternary centers. Chiral enamine auxiliaries bearing 9‐anthryl substituents were found to confer the highest levels of stereoinduction.
Discussion Addendum For: Protection of Alcohols Using 2‐Benzyloxy‐1‐Methylpyridinium Trifluoromethanesulfonate: Methyl (
R
)‐(‐)‐3‐Benzyloxy‐2‐methyl Propanoate
Authors: Fulo Harvey F. , Albiniak Philip A. , Dudley* Gregory B.
Published: April 2020

Benzyl ethers are important protecting groups in organic synthesis, but traditional installation methods, such as the Williamson ether synthesis or reaction of an alcohol with benzyl trichloroacetimidate, often limit their formation with substrates containing functional groups unable to tolerate strongly basic or acidic conditions, respectively. The reaction of N,N‐dimethylaniline occurred via an N‐methyl to benzyl exchange instead of the expected electrophilic aromatic substitution. This illustrates that amines can also trap the putative phenylcarbenium species released during the thermal activation of 2‐benzyloxy‐1‐methylpyridinium triflate (BnOPT). Benzylation can generally be achieved chemoselectively using BnOPT with triethylamine in the presence of alcohols, amides, and other functional groups. The mild and nearly neutral conditions associated with the use of BnOPT is the main advantage of the benzylation protocol.
Discussion Addendum for: Preparation of Enantioenriched Homoallylic Primary Amines
Authors: Yus* Miguel
Published: April 2020

One of the most efficient methodologies to prepare amines is the addition of organometallic reagents to imines. From a synthetic point of view, the corresponding allylation presents special interest due to the possible transformation of the introduced carbon‐carbon double bond into other functionalities. This addendum discusses the preparation of enantioenriched homoallylic primary amines. The most commonly used method to prepare the starting enantiomerically pure
N‐tert
‐butylsulfinyl imines is the direct reaction of the corresponding carbonyl compounds with enantiomerically pure commercially available
tert
‐butanesulfinamides and titanium tetraethoxide under thermal or microwave activation. The addendum also provides information on the synthetic applications of chiral N‐sulfinyl homoallylic amines.
Discussion Addendum for: Synthesis of Ynamides by Copper‐Mediated Coupling of 1,1‐Dibromo‐1‐Alkenes with Nitrogen Nucleophiles. Preparation of 4‐Methyl‐N‐(2‐Phenylethynyl)‐N‐(Phenylmethyl)Benzenesulfonamide
Authors: Theunissen Cédric , Thilmany Pierre , Lahboubi Mounsef , Blanchard Nicolas , Evano* Gwilherm
Published: April 2020

Ynamides provide unique opportunities not only for the introduction of nitrogen‐containing motifs into organic molecules but also for the generation of otherwise hardly accessible highly reactive intermediates. This addendum discusses the synthesis of ynamides with copper as the general solution. It provides information on 1,1‐dibromo‐1‐alkenes as practical alkynylating agents. The addendum also discusses the chemistry of ynamides, which is a versatile and useful building blocks in organic synthesis. The development of efficient synthetic procedures for the preparation of ynamides, including those from 1,1‐dibromo‐1‐alkenes, paved the way for their use as building blocks in organic chemistry. Besides the use of strong acids, electrophilic metal complexes have also proven successful to activate ynamides.