bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the domain structure and the DNA binding site of the transcriptional activator FhlA
Authors: Leonhartsberger Susanne , Ehrenreich Armin , Böck August
Published: March 2003

FhlA is the transcriptional activator of the genes coding for the formate hydrogen lyase system in Escherichia coli. It is activated by the binding of formate and induces transcription by σ54 RNA polymerase after binding to specific upstream activating sequences (UAS). Sequence comparison had shown that FhlA exhibits a structure composed of three domains, which is typical for σ54‐dependent regulators. By analyzing the N‐terminal domain of FhlA of E. coli (amino acids 1–378; FhlA‐N) and the rest of the protein (amino acids 379–693; FhlA‐C) as separate proteins in vivo and in vitro the functions of the different domains of FhlA were elucidated. The FhlA‐C domain is active in ATP hydrolysis and activation of transcription and its activity is neither influenced by the presence of formate nor of the antiactivator HycA. However, it is stimulated in the presence of the FhlA‐specific UAS, indicating that this region of FhlA is responsible for DNA binding. FhlA‐N is not active itself but able to reduce the activity of full‐length FhlA in trans, probably by formation of nonfunctional heterooligomers. The DNA binding site of FhlA was analyzed by hydroxyradical footprinting. Each UAS consists of two binding sites of 16 bp separated by a spacer region. A consensus sequence could be deduced and a model is presented and supported by in vivo data in which a FhlA tetramer binds to the UAS on one side of the DNA helix. Performing an extensive screening we could show that the FhlA regulatory system is conserved in different species of the family Enterobacteriaceae. The analysis of orthologs of FhlA revealed that they are able to functionally replace the E. coli enzyme.
The pro‐ or anti‐apoptotic function of NF‐κB is determined by the nature of the apoptotic stimulus
Authors: Kaltschmidt Barbara , Kaltschmidt Christian , Hofmann Thomas G. , Hehner Steffen P. , Dröge Wulf , Schmitz M. Lienhard
Published: March 2003

To test whether the behaviour of transcription factor NF‐κB as a promoter or antagonist of apoptosis depends on the apoptotic stimulus, we determined the influence of NF‐κB on cell killing elicited by a variety of inducers within a given cell type. Inhibition of NF‐κB by genetic and pharmacological approaches rendered HeLa cells more susceptible to TNF‐α‐induced cell killing, but protected them almost completely from H2O2‐ and pervanadate‐induced apoptosis. TNF‐α was unable to protect HeLa from H2O2‐ and pervanadate‐induced apoptosis and further enhanced the cytotoxicity induced by these two adverse agents. Supernatants from HeLa cells stably overexpressing a transdominant negative form of IκB‐α selectively increased the cytotoxicity of TNF‐α for HeLa cells, suggesting that the enhanced susceptibility of these cells can be attributed to one or more secretable factors. Supershift experiments showed that the various apoptotic stimuli induced the same subset of DNA‐binding subunits. Therefore, the nature of the signals elicited by the respective death inducers determines whether NF‐κB induction leads to apoptosis or survival, suggesting that the manipulation of NF‐κB activity may provide a new approach to adjuvant therapy in cancer treatment.
Structure and location of a ferritin gene of the yellow fever mosquito Aedes aegypti
Authors: Pham Daphne Q.‐D. , Brown Susan E. , Knudson Dennis L. , Winzerling Joy J. , Dodson Mark S. , Shaffer James J.
Published: March 2003

We have isolated and sequenced a genomic clone encoding the 24‐ and 26‐kDa ferritin subunits in the mosquito Aedes aegypti (Rockefeller strain). The A. aegypti gene differs from other known ferritin genes in that it possesses an additional intron and an unusually large second intron. The additional intron is located within the 5′ untranslated region, between the CAP site and the start codon. The second intron contains numerous putative transposable elements. In addition, unlike the human and rat ferritin genes, the A. aegypti ferritin gene is a single copy gene, located at 88.3% FLpter on the q‐arm of chromosome 1. Primer extension analysis indicates that the A. aegypti ferritin gene has multiple transcriptional start sites. A differential usage of these sites is observed with varied cellular iron concentrations.
Identification of a nuclear export signal in MKK6, an activator of the carp p38 mitogen‐activated protein kinases
Authors: Hashimoto Hisashi , Fukuda Makoto , Matsuo Yoshiyuki , Yokoyama Yoshihiro , Nishida Eisuke , Toyohara Haruhiko , Sakaguchi Morihiko
Published: March 2003

Carp homologues of p38 mitogen‐activated protein kinase (MAPK) and its activator MAPK kinase 6 (MAPKK6, referred to as MKK6) were identified. There exist at least two distinct carp p38s, cp38a and cp38b, both of which consist of 361 amino acids. The transcript of c38a was exclusively expressed in the ovary, whereas that of cp38b was ubiquitously expressed. Western blot analysis with anti‐(phosphorylated MAPK) Ig specific to the active p38 or JNK has shown that p38 was activated in response to hypertonic stress (1 m sorbitol) in epithelioma papilosum cyprini carp epithelial cells (EPC) and that the activation of p38 proceeded faster to the maximal level than that of JNK. Carp homologue (cMKK6) of p38 activator MKK6 consists of 404 amino acids. It was expressed ubiquitously but was most abundant in the ovary. An in vitro kinase assay demonstrated that cMKK6 is an upstream activator of cp38 and cp38b in carp because it specifically phosphorylated and activated cp38a and cp38b. Interestingly, we found that cMKK6 has a nuclear export signal (NES) sequence in its N‐terminal region although upstream activators of stress‐activated MAPKs, p38 and JNK, do not in other animals. The NES sequence facilitated nuclear export of cMKK6 and ovalbumin. Leucine residues in the sequence were crucial for the NES activity, as the activity was lost on replacement of the leucines to alanines. The existence of an NES in cMKK6 implies the requisite of strict regulation of the p38 MAPK pathway in carp. The abundance of these components for the stress‐activated pathway in the ovary might be related to ectogenetic early development.
Increased methyl esterification of altered aspartyl residues in erythrocyte membrane proteins in response to oxidative stress
Authors: Ingrosso Diego , D’Angelo Stefania , di Carlo Enza , Perna Alessandra F. , Zappia Vincenzo , Galletti Patrizia
Published: March 2003

Protein‐l‐isoaspartate (d‐aspartate) O‐methyltransferase (PCMT; EC 2.1.1.77) catalyses the methyl esterification of the free α‐carboxyl group of abnormal l‐isoaspartyl residues, which occur spontaneously in protein and peptide substrates as a consequence of molecular ageing. The biological function of this transmethylation reaction is related to the repair or degradation of age‐damaged proteins. Methyl ester formation in erythrocyte membrane proteins has also been used as a marker reaction to tag these abnormal residues and to monitor their increase associated with erythrocyte ageing diseases, such as hereditary spherocytosis, or cell stress (thermal or osmotic) conditions.

The study shows that levels of l‐isoaspartyl residues rise in membrane proteins of human erythrocytes exposed to oxidative stress, induced by t‐butyl hydroperoxide or H2O2. The increase in malondialdehyde content confirmed that the cell membrane is a primary target of oxidative alterations. A parallel rise in the methaemoglobin content indicates that proteins are heavily affected by the molecular alterations induced by oxidative treatments in erythrocytes. Antioxidants largely prevented the increase in membrane protein methylation, underscoring the specificity of the effect. Conversely, we found that PCMT activity, consistent with its repair function, remained remarkably stable under oxidative conditions, while damaged membrane protein substrates increased significantly. The latter include ankyrin, band 4.1 and 4.2, and the integral membrane protein band 3 (the anion exchanger). The main target was found to be particularly protein 4.1, a crucial element in the maintenance of membrane‐cytoskeleton network stability. We conclude that the increased formation/exposure of l‐isoaspartyl residues is one of the major structural alterations occurring in erythrocyte membrane proteins as a result of an oxidative stress event. In the light of these and previous findings, the occurrence of isoaspartyl sites in membrane proteins as a key event in erythrocyte spleen conditioning and hemocatheresis is proposed.
Proteomic analysis of differential protein expression in primary hepatocytes induced by EGF, tumour necrosis factor α or the peroxisome proliferator nafenopin
Authors: Chevalier Stephan , Macdonald Neil , Tonge Robert , Rayner Steve , Rowlinson Rachel , Shaw Joanne , Young Janice , Davison Matthew , Roberts Ruth A.
Published: March 2003

Peroxisome proliferators are nongenotoxic rodent‐liver carcinogens that have been shown to cause both an induction of hepatocyte proliferation and a suppression of apoptosis. Both epidermal growth factor (EGF) and the peroxisome proliferator nafenopin induce DNA replication in primary rat hepatocyte cultures, but apparently through different signalling pathways. However, both EGF and nafenopin require tumour necrosis factor α (TNFα) signalling to induce DNA replication. By examining proteins isolated from rat primary hepatocyte cultures using two‐dimensional gel electrophoresis and mass spectrometry, we found that proteins showing an altered expression pattern in response to nafenopin differed from those showing altered expression in response to EGF. However, many proteins showing altered expression upon stimulation with TNFα were common to both the EGF and nafenopin responses. These proteome profiling experiments contribute to a better understanding of the molecular mechanisms involved in the response to peroxisome proliferators. We found 32 proteins with altered expression upon stimulation with nafenopin, including muscarinic acetylcholine receptor 3, intermediate filament vimentin and the β subunit of the ATP synthase. These nonperoxisomal protein targets offer insights into the mechanisms of peroxisome proliferator‐induced carcinogenesis in rodents and provide opportunities to identify toxicological markers to facilitate early identification of nongenotoxic carcinogens.
Families of retinoid dehydrogenases regulating vitamin A function
Authors: Duester Gregg
Published: March 2003

Vitamin A (retinol) and provitamin A (β‐carotene) are metabolized to specific retinoid derivatives which function in either vision or growth and development. The metabolite 11‐cis‐retinal functions in light absorption for vision in chordate and nonchordate animals, whereas all‐trans‐retinoic acid and 9‐cis‐retinoic acid function as ligands for nuclear retinoic acid receptors that regulate gene expression only in chordate animals. Investigation of retinoid metabolic pathways has resulted in the identification of numerous retinoid dehydrogenases that potentially contribute to metabolism of various retinoid isomers to produce active forms. These enzymes fall into three major families. Dehydrogenases catalyzing the reversible oxidation/reduction of retinol and retinal are members of either the alcohol dehydrogenase (ADH) or short‐chain dehydrogenase/reductase (SDR) enzyme families, whereas dehydrogenases catalyzing the oxidation of retinal to retinoic acid are members of the aldehyde dehydrogenase (ALDH) family. Compilation of the known retinoid dehydrogenases indicates the existence of 17 nonorthologous forms: five ADHs, eight SDRs, and four ALDHs, eight of which are conserved in both mouse and human. Genetic studies indicate in vivo roles for two ADHs (ADH1 and ADH4), one SDR (RDH5), and two ALDHs (ALDH1 and RALDH2) all of which are conserved between humans and rodents. For several SDRs (RoDH1, RoDH4, CRAD1, and CRAD2) androgens rather than retinoids are the predominant substrates suggesting a function in androgen metabolism as well as retinoid metabolism.
Ca2+ signalling and muscle disease
Authors: MacLennan David H.
Published: March 2003

Transient elevations of intracellular Ca2+ play a signalling role in such complex cellular functions as contraction, secretion, fertilization, proliferation, metabolism, heartbeat and memory. However, prolonged elevation of Ca2+ above about 10 µm is deleterious to a cell and can activate apoptosis. In muscle, there is a narrow window of Ca2+ dysregulation in which abnormalities in Ca2+ regulatory proteins can lead to disease, rather than apoptosis. Key proteins in the regulation of muscle Ca2+ are the voltage‐dependent, dihydropyridine‐sensitive, l‐type Ca2+ channels located in the transverse tubule and Ca2+ release channels in the junctional terminal cisternae of the sarcoplasmic reticulum. Abnormalities in these proteins play a key role in malignant hyperthermia (MH), a toxic response to anesthetics, and in central core disease (CCD), a muscle myopathy. Sarco(endo)plasmic reticulum Ca2+ ATPases (SERCAs) return sarcoplasmic Ca2+ to the lumen of the sarcoplasmic reticulum. Loss of SERCA1a Ca2+ pump function is one cause of exercise‐induced impairment of the relaxation of skeletal muscle, in Brody disease. Phospholamban expressed in cardiac muscle and sarcolipin expressed in skeletal muscle regulate SERCA activity. Studies with knockout and transgenic mice show that gain of inhibitory function of phospholamban alters cardiac contractility and could be a causal feature in some cardiomyopathies. Calsequestrin, calreticulin, and a series of other acidic, lumenal, Ca2+ binding proteins provide a buffer for Ca2+ stored in the sarcoplasmic reticulum. Overexpression of cardiac calsequestrin leads to cardiomyopathy and ablation of calreticulin alters cardiac development.
Roles of cysteines in rat dipeptidyl peptidase IV/CD26 in processing and proteolytic activity
Authors: Dobers Jörg , Grams Sabine , Reutter Werner , Fan Hua
Published: March 2003

The multifunctional type II transmembrane glycoprotein, dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5), is expressed by almost all mammalian cells and is identical to the adenosine deaminase binding protein CD26 on lymphocytes. The extracellular part of rat DPPIV can be divided into three domains the middle part of which harbors 10 of the 12 highly conserved cysteine residues. The cysteine‐rich domain is responsible for DPPIV‐binding to collagen I and to extracellular ADA. The participation of distinct cysteines in disulfide bridges is not yet known. Titration experiments have shown the presence of six free cysteines and three disulfide bridges in native rat DPPIV. To investigate the role of distinct cysteines in the structure–function relationships of rat DPPIV we constructed 12 different cysteine point mutations (C299, C326, C383, C455, C650 mutated to G; C337, C395, C445, C448, C473, C552, C763 mutated to S). Intracellular translocation to the cell surface of stable transfected Chinese hamster ovary cells was examined with antibodies against different epitopes of DPPIV. Surface expression of mutants C326G, C445S and C448S is inhibited totally; mutants C337S, C455G, C473S and C552S show weak expression only. In parallel, the half‐life of these mutants is reduced to < 10% compared with wild‐type enzyme. We were able to show that the specific peptidase activity of the mutant protein depends on cell‐surface expression, dimerization and the existence of a 150‐kDa form demonstrable by nondenaturing SDS/PAGE. We conclude that cysteine residues 326, 337, 445, 448, 455, 473 and 552 in rat DPPIV are essential for the correct folding and intracellular trafficking of this glycoprotein, and therefore for its normal biological properties.
Control of cellular phosphatidylinositol 4,5‐bisphosphate levels by adhesion signals and Rho GTPases in NIH 3T3 fibroblasts
Authors: Weernink Paschal A. Oude , Guo Yuanjian , Zhang Chunyi , Schmidt Martina , von Eichel‐Streiber Christoph , Jakobs Karl H.
Published: March 2003

The involvement of small GTPases of the Rho family in the control of phosphoinositide metabolism by adhesion signals was examined in NIH 3T3 fibroblasts. Abrogation of adhesion signals by detachment of cells from their substratum resulted in a time‐dependent decrease in the cellular level of PtdIns(4,5)P2 by ≈ 50%. This effect could be mimicked by treatment of adherent cells with Clostridium difficile toxin B and toxin B‐1470, which inhibit specific subsets of Rho and Ras GTPases. Detachment of cells that had been pretreated with the clostridial toxins did not cause a further reduction in PtdIns(4,5)P2 levels, suggesting that the target GTPases are integrated into the control of phosphoinositide levels by adhesion signals. The reduction in PtdIns(4,5)P2 levels could be attributed to reduced activity of the major PtdIns(4,5)P2‐producing enzyme, PtdIns4P 5‐kinase. Unexpectedly, both cell detachment and toxin treatment resulted in a twofold to threefold increase in inositol phosphate production in intact cells. In lysates of these cells, in vitro phospholipase C activity was found to be elevated by 30–50%. The effects of cell detachment and toxin treatment on inositol phosphate formation could be mimicked by expression of dominant‐negative N17 Rac1. Taken together, these data suggest that adhesion‐controlled small GTPases of the Rho family are involved in the regulation of the cellular PtdIns(4,5)P2 levels in NIH 3T3 fibroblasts, by controlling the activities of both PtdIns4P 5‐kinase and phospholipase C.
Structure elucidation and biological activity of an unusual adipokinetic hormone from corpora cardiaca of the butterfly, Vanessa cardui
Authors: Köllisch Gabriele V. , Lorenz Matthias W. , Kellner Roland , Verhaert Peter D. , Hoffmann Klaus H.
Published: March 2003

A structurally unusual member of the adipokinetic hormone/red pigment‐concentrating hormone peptide family was isolated from corpora cardiaca of the painted lady butterfly, Vanessa cardui. Its primary structure was assigned by Edman degradation and nano‐electrospray‐time‐of‐flight mass spectrometry as pQLTFTSSWGGK (Vac‐AKH). Vac‐AKH represents the first 11mer and the first nonamidated peptide in this family.

The peptide shows significant adipokinetic activity in adult specimens of V. cardui. Injection of 10 pmol of synthetic Vac‐AKH into 4‐day‐old decapitated males resulted in an ≈ 150% increase of hemolymph lipids after 90 min. Half maximal adipokinetic activity was achieved with about 0.1 pmol of Vac‐AKH. During a 2‐h incubation of corpora cardiaca/corpora allata complexes in medium containing 50 mm KCl, significant amounts of Vac‐AKH were released from the glands.
Endoglin expression on human microvascular endothelial cells
Authors: Wong S. H. , Hamel L. , Chevalier S. , Philip A.
Published: March 2003

Transforming growth factor‐β (TGF‐β) plays an important role in angiogenesis and vascular function. Endoglin, a transmembrane TGF‐β binding protein, is highly expressed on vascular endothelial cells and is the target gene for the hereditary haemorrhagic telangiectasia type I (HHT1), a dominantly inherited vascular disorder. The specific function of endoglin responsible for HHT1 is believed to involve alterations in TGF‐β responses. The initial interactions on the cell surface between endoglin and TGF‐β receptors may be an important mechanism by which endoglin modulates TGF‐β signalling, and thereby responses. Here it is shown that on human microvascular endothelial cells, endoglin is co‐expressed and is associated with betaglycan, a TGF‐β accessory receptor with which endoglin shares limited amino acid homology. This complex formation may occur in either a ligand‐dependent or a ligand‐independent manner. In addition, the occurrence of three higher order complexes containing endoglin, type II and/or type I TGF‐β receptors, on these cells is demonstrated. Our findings suggest that endoglin may modify TGF‐β signalling by interacting with both betaglycan and the TGF‐β signalling receptors at physiological receptor concentrations and ratios.
Episomal vectors for gene expression in mammalian cells
Authors: Van Craenenbroeck Kathleen , Vanhoenacker Peter , Haegeman Guy
Published: March 2003

An important reason for preferring mammalian cells for heterologous gene expression is their ability to make authentic proteins containing post‐translational modifications similar to those of the native protein. The development of expression systems for mammalian cells has been ongoing for several years, resulting in a wide variety of effective expression vectors. The aim of this review is to highlight episomal expression vectors. Such episomal plasmids are usually based on sequences from DNA viruses, such as BK virus, bovine papilloma virus 1 and Epstein–Barr virus. In this review we will mainly focus on the improvements made towards the usefulness of these systems for gene expression studies and gene therapy.
Isolation, synthesis and pharmacological characterization of δ‐palutoxins IT, novel insecticidal toxins from the spider Paracoelotes luctuosus (Amaurobiidae)
Authors: Corzo Gerardo , Escoubas Pierre , Stankiewicz Maria , Pelhate Marcel , Kristensen Charles P. , Nakajima Terumi
Published: March 2003

Four novel insecticidal toxins were isolated from the venom of the spider Paracoelotes luctuosus (Araneae: Amaurobiidae) and named δ‐palutoxins IT1 to IT4. The four toxins are homologous 36–37 amino acid peptides reticulated by four disulfide bridges and three have amidated C‐terminal residues. The δ‐palutoxins are highly homologous with the previously described µ‐agatoxins and curtatoxins (77–97%). The four peptides demonstrated significant toxicity against larvae of the crop pest Spodoptera litura (Lepidoptera: Noctuidae) in a microinjection bioassay, with LD50 values in the 9–50 µg per g of insect range. This level of toxicity is equivalent to that of several of the most active scorpion toxins used in the development of recombinant baculoviruses, and the δ‐palutoxins appear to be insect specific. Electrophysiological experiments demonstrated that δ‐palutoxin IT1, the most active toxin acts by affecting insect sodium channel inactivation, resulting in the appearance of a late‐maintained sodium current, in a similar fashion to insecticidal scorpion α and α‐like toxins and is thus likely to bind to channel receptor site 3. However, δ‐palutoxin IT1 was distinguished by its lack of effect on peak sodium conductance, on the early phase of sodium current inactivation and the absence of a shift in the activation voltage of the sodium channels. δ‐Palutoxins are thus proposed as new insecticidal toxins related to the α and α‐like scorpion toxins. They will be useful both in the development of recombinant baculoviruses in agrochemical applications and also as molecular probes for the investigation of molecular mechanisms of insect selectivity and structure and function of sodium channels.
Substrate and solvent isotope effects on the fate of the active oxygen species in substrate‐modulated reactions of putidamonooxin
Authors: Twilfer Hans , Sandfort Georg , Bernhardt Frithjof‐Hans
Published: March 2003

Using 4‐methoxybenzoate monooxygenase from Pseudomonas putida, the substrate deuterium isotope effect on product formation and the solvent isotope effect on the stoichiometry of oxygen uptake, NADH oxidation, product and/or H2O2 (D2O2) formation for tight couplers, partial uncouplers, and uncouplers as substrates were measured. These studies revealed for the true, intrinsic substrate deuterium isotope effect on the oxygenation reaction a k1H/k2H ratio of < 2.0, derived from the inter‐ and intramolecular substrate isotope effects. This value favours a concerted oxygenation mechanism of the substrate. Deuterium substitution in a tightly coupling substrate initiated a partial uncoupling of oxygen reduction and substrate oxygenation, with release of H2O2 corresponding to 20% of the overall oxygen uptake. This H2O2 (D2O2) formation (oxidase reaction) almost completely disappeared when the oxygenase function was increased by deuterium substitution in the solvent. The electron transfer from NADH to oxygen, however, was not affected by deuterium substitution in the substrate and/or the solvent.

With 4‐trifluoromethylbenzoate as uncoupling substrate and D2O as solvent, a reduction (peroxidase reaction) of the active oxygen complex was initiated in consequence of its extended lifetime. These additional two electron‐transfer reactions to the active oxygen complex were accompanied by a decrease of both NADH oxidation and oxygen uptake rates. These findings lead to the following conclusions: (a) under tightly coupling conditions the rate‐limiting step must be the formation time and lifetime of an active transient intermediate within the ternary complex iron/peroxo/substrate, rather than an oxygenative attack on a suitable C–H bond or electron transfer from NADH to oxygen. Water is released after the monooxygenation reaction; (b) under uncoupling conditions there is competition in the detoxification of the active oxygen complex between its protonation (deuteronation), with formation of H2O2 (D2O2) and its further reduction to water. The additional two electron‐transfer reactions onto the active oxygen complex then become rate limiting for the oxygen uptake rate.
Polyamines as gating molecules of inward‐rectifier K+ channels
Authors: Oliver Dominik , Baukrowitz Thomas , Fakler Bernd
Published: March 2003

Inward‐rectifier potassium (Kir) channels comprise a superfamily of potassium (K+) channels with unique structural and functional properties. Expressed in virtually all types of cells they are responsible for setting the resting membrane potential, controlling the excitation threshold and secreting K+ ions. All Kir channels present an inwardly rectifying current–voltage relation, meaning that at any given driving force the inward flow of K+ ions exceeds the outward flow for the opposite driving force. This inward‐rectification is due to a voltage‐dependent block of the channel pore by intracellular polyamines and magnesium. The present molecular–biophysical understanding of inward‐rectification and its physiological consequences is the topic of this review. In addition to polyamines, Kir channels are gated by intracellular protons, G‐proteins, ATP and phospholipids depending on the respective Kir subfamily as detailed in the following review articles.
G‐protein mediated gating of inward‐rectifier K+ channels
Authors: Mark Melanie D. , Herlitze Stefan
Published: March 2003

G‐protein regulated inward‐rectifier potassium channels (GIRK) are part of a superfamily of inward‐rectifier K+ channels which includes seven family members. To date four GIRK subunits, designated GIRK1–4 (also designated Kir3.1–4), have been identified in mammals, and GIRK5 has been found in Xenopus oocytes. GIRK channels exist in vivo both as homotetramers and heterotetramers. In contrast to the other mammalian GIRK family members, GIRK1 can not form functional channels by itself and has to assemble with GIRK2, 3 or 4. As the name implies, GIRK channels are modulated by G‐proteins; they are also modulated by phosphatidylinositol 4,5‐bisphosphate, intracellular sodium, ethanol and mechanical stretch. Recently a family of GTPase activating proteins known as regulators of G‐protein signaling were shown to be the missing link for the fast deactivation kinetics of GIRK channels in native cells, which contrast with the slow kinetics observed in heterologously expressed channels. GIRK1, 2 and 3 are highly abundant in brain, while GIRK4 has limited distribution. Here, GIRK1/2 seems to be the predominant heterotetramer. In general, neuronal GIRK channels are involved in the regulation of the excitability of neurons and may contribute to the resting potential. Interestingly, only the GIRK1 and 4 subunits are distributed in the atrial and sinoatrial node cells of the heart and are involved in the regulation of cardiac rate. Our main objective of this review is to assess the current understanding of the G‐protein modulation of GIRK channels and their physiological importance in mammals.
Insect chitin synthase
Authors: Tellam Ross L. , Vuocolo Tony , Johnson Samuel E. , Jarmey Janine , Pearson Roger D.
Published: March 2003

Chitin is a major component of the cuticle of arthropods. However, the synthesis of chitin is poorly understood. Feeding larvae of the insect Lucilia cuprina on the fungal chitin synthase competitive inhibitor, nikkomycin Z resulted in strong concentration‐dependent mortality of the larvae (LD50 = 280 nm). This result demonstrates that chitin is an essential component of this insect. The complete cDNA and deduced amino‐acid sequences of the first arthropod chitin synthase‐like protein, LcCS‐1, from the larvae of the insect L. cuprina have been determined. The cDNA sequence is 5757 bp in length and codes for a large complex protein containing 1592 amino acids (Mr = 180 717). Analysis of the whole protein sequence reveals low, but significant, similarity to yeast chitin synthases with stronger areas of conservation centred on local regions implicated in the active sites of the yeast enzymes. Strikingly, LcCS‐1 contains 15–18 potential transmembrane segments, indicating that the protein is an integral membrane protein. Two alternative topographical models of LcCS‐1 are described, which involve its association with either the plasma membrane or the membrane of intracellular vesicles. LcCS‐1 mRNA is produced in all life stages of the insect with expression in the larval stage limited to the integument and trachea. In a third instar larva the mRNA was localized to a single layer of epidermal cells immediately underlying the procuticle region of the integument. cDNA or genomic sequences that are highly related to fragments of LcCS‐1 were demonstrated in three insect orders, one arachnid and Caenorhabditis elegans, thereby attesting to the importance of this enzyme in these chitin‐producing organisms. Bioinformatics has been used to deduce the gene sequence and organization of the highly homologous Drosophila melanogaster orthologue of LcCS‐1, DmCS‐1.
Evidence for an amphipathicity independent cellular uptake of amphipathic cell‐penetrating peptides
Authors: Scheller Anne , Wiesner Burkhard , Melzig Matthias , Bienert Michael , Oehlke Johannes
Published: March 2003

The cellular uptake of a peptide set derived from membrane‐permeable α‐helical amphipathic peptides by stepwise alterations of structure forming propensity and charge was studied by confocal laser scanning microscopy (CLSM) combined with HPLC. For CLSM monitoring, an online protocol was employed that avoided bias of the uptake results by washout. Using this protocol, extensive fluorescence, approaching the intensity of the external peptide, was observed in the cytosol and nucleus within minutes in all cases, irrespective of the degree of amphipathicity. HPLC analyses of the cell lysates revealed the unmetabolized peptides to be the predominant source of the intracellular fluorescence. Significant amphipathicity‐dependent differences became apparent only after washing the peptide‐loaded cells, reflecting the effects of amphipathicity on resistance to wash out. Exposure of the cells to the peptides at 37 and 0 °C led to similar results, indicating the nonendocytic character of the uptake.

With a view to practical applications , the results of the present study open the possibility of exploiting nonamphipathic peptides as vectors for translocating polar compounds into the cell interior, which would circumvent substantial obstacles currently connected with the use of amphipathic vector peptides, such as membrane toxicity and low solubility. Moreover, differences in the uptake of several members of the investigated peptide series into different cell types present a promising basis for the design of cell‐type specific vector peptides.
Structure–function relationships in spruce budworm antifreeze protein revealed by isoform diversity
Authors: Doucet Daniel , Tyshenko Michael G. , Kuiper Michael J. , Graether Steffen P. , Sykes Brian D. , Daugulis Andrew J. , Davies Peter L. , Walker Virginia K.
Published: March 2003

The spruce budworm, Choristoneura fumiferana, produces antifreeze protein (AFP) to assist in the protection of the overwintering larval stage. AFPs are thought to lower the freezing point of the hemolymph, noncolligatively, by interaction with the surface of ice crystals. Previously, we had identified a cDNA encoding a 9‐kDa AFP with 10–30 times the thermal hysteresis activity, on a molar basis, than that shown by fish AFPs. To identify important residues for ice interaction and to investigate the basis for the hyperactivity of the insect AFPs, six new spruce budworm AFP cDNA isoforms were isolated and sequenced. They differ in amino‐acid identity as much as 36% from the originally characterized AFP and can be divided into three classes according to the length of their 3′ untranslated regions (UTRs). The new isoforms have at least five putative ‘Thr‐X‐Thr’ ice‐binding motifs and three of the new isoforms encode larger, 12‐kDa proteins. These appear to be a result of a 30 amino‐acid insertion bearing two additional ice‐binding motifs spaced 15 residues apart. Molecular modeling, based on the NMR structure of a short isoform, suggests that the insertion folds into two additional β‐helix loops with their Thr‐X‐Thr motifs in perfect alignment with the others. The first Thr of the motifs are often substituted by Val, Ile or Arg and a recombinantly expressed isoform with both Val and Arg substitutions, showed wild‐type thermal hysteresis activity. The analysis of these AFP isoforms suggests therefore that specific substitutions at the first Thr in the ice binding motif can be tolerated, and have no discernible effect on activity, but the second Thr appears to be conserved. The second Thr is thus likely important for the dynamics of initial ice contact and interaction by these hyperactive antifreezes.
Thioredoxin reductase as a pathophysiological factor and drug target
Authors: Becker Katja , Gromer Stephan , Schirmer R. Heiner , Müller Sylke
Published: March 2003

Human cytosolic thioredoxin reductase (TrxR), a homodimeric protein containing 1 selenocysteine and 1 FAD per subunit of 55 kDa, catalyses the NADPH‐dependent reduction of thioredoxin disulfide and of numerous other oxidized cell constituents. As a general reducing enzyme with little substrate specificity, it also contributes to redox homeostasis and is involved in prevention, intervention and repair of damage caused by H2O2‐based oxidative stress.

Being a selenite‐reducing enzyme as well as a selenol‐containing enzyme, human TrxR plays a central role in selenium (patho)physiology. Both dietary selenium deficiency and selenium oversupplementation, a lifestyle phenomenon of our time, appear to interfere with the activity of TrxR. Selenocysteine 496 of human TrxR is a major target of the anti‐rheumatic gold‐containing drug auranofin, the formal Ki for the stoichiometric inhibition being 4 nm. The hypothesis that TrxR and extracellular thioredoxin play a pathophysiologic role in chronic diseases such as rheumatoid arthritis, Sjögren's syndrom, AIDS, and certain malignancies, is substantiated by biochemical, virological, and clinical evidence. Reduced thioredoxin acts as an autocrine growth factor in various tumour diseases, as a chemoattractant, and it synergises with interleukins 1 and 2. The effects of anti‐tumour drugs such as carmustine and cisplatin can be explained in part by the inhibition of TrxR. Consistently, high levels of the enzyme can support drug resistance.

TrxRs from different organisms such as Escherichia coli, Mycobacterium leprae, Plasmodium falciparum, Drosophila melanogaster, and man show a surprising diversity in their chemical mechanism of thioredoxin reduction. This is the basis for attempts to develop specific TrxR inhibitors as drugs against bacterial infections like leprosy and parasitic diseases like amebiasis and malaria.
Signal transduction pathways triggered by fibroblast growth factor receptor 1 expressed in Xenopus laevis oocytes after fibroblast growth factor 1 addition
Authors: Browaeys‐Poly Edith , Cailliau Katia , Vilain Jean‐Pierre
Published: March 2003

Xenopus oocytes expressing fibroblast growth factor receptor 1 (FGFR1) were used as a biological model system to analyse the signal transduction pathways that are triggered by fibroblast growth factor 1 (FGF1). Germinal vesicle breakdown (GVBD) and phosphorylation of extracellular signal‐regulated protein kinase 2 (ERK2) occured 15 h after FGF1 addition. These events were Ras‐dependent as they were blocked by a Ras dominant negative form. The Ras activity was promoted by three upstream effectors, growth factor‐bound protein 2 (Grb2), phosphatidylinositol 3‐kinase (PI3K) and Src cytoplasmic kinase. Ras activation was inhibited by a Grb2 dominant negative form (P49L), by PI3K inhibitors, including wortmannin, LY294002, the N‐SH2 domain of p85α PI3K and by the SH2 domain of Src. Src activation induced by FGF1 was blocked by the SH2 domain of Src and PP2, a specific inhibitor of Src. The Grb2 adaptor was recruited by the upstream Src homology 2/α‐collagen‐related (Shc) effector, as the SH2‐Shc domain prevented the GVBD and the ERK2 phosphorylation induced by FGF1. The importance of another signalling pathway involving phospholipase Cγ (PLCγ) was also investigated. The use of the PLCγ inhibitory peptide, neomycin and the calcium chelator BAPTA‐AM on oocytes expressing FGFR1 or the stimulation by PDGF‐BB of oocytes expressing PDGFR‐FGFR1 mutated on the PLCγ binding site, prevented GVBD and ERK2 phosphorylation. This study shows that the transduction cascade induced by the FGFR1–FGF1 interaction in Xenopus oocytes represents the sum of Ras‐dependent and PLCγ‐dependent pathways. It emphasizes the role played by PI3K and Src and their connections with the Ras cascade in the FGFR1 signal transduction.
Characterization of the internal motions of a chimeric protein by 13C NMR highlights the important dynamic consequences of the engineering on a millisecond time scale
Authors: Wolff Nicolas , Guenneugues Marc , Gilquin Bernard , Drakopoulou Eugenia , Vita Claudio , Ménez André , Zinn‐Justin Sophie
Published: March 2003

By transferring the central curaremimetic β hairpin of the snake toxin α into the scaffold of the scorpion charybdotoxin, a chimeric protein was constructed that reproduced the three‐dimensional structure and partially reproduced the function of the parent β hairpin, without perturbing the three‐dimensional structure of the scaffold [1]. Picosecond to hour time scale motions of charybdotoxin and the engineered protein were observed, in order to evaluate the dynamic consequences of the six deletions and eight mutations differentiating the two molecules. The chimeric protein dynamics were also compared to that of toxin α, in order to examine the β hairpin motions in both structural contexts. Thus, 13C R1, R1ρ and 1H→13C nOe were measured for all the CαHα and threonine CβHβ vectors. As the proteins were not labeled, accordion techniques combined to coherence selection by pulsed field gradients and preservation of magnetization following equivalent pathways were used to considerably reduce the spectrometer time needed. On one hand, we observed that the chimeric protein and charybdotoxin are subjected to similar picosecond to nanosecond time scale motions except around the modified β sheet region. The chimeric protein also exhibits an additional millisecond time scale motion on its whole sequence, and its β structure is less stable on a minute to hour time scale. On the other hand, when the β hairpin dynamics is compared in two different structural contexts, i.e. in the chimeric protein and the curaremimetic toxin α, the picosecond to nanosecond time scale motions are fairly conserved. However, the microsecond to millisecond time scale motions are different on most of the β hairpin sequence, and the β sheet seems more stable in toxin α than in the chimera. The slower microsecond to hour time scale motions seem to be extremely sensitive to the structural context, and thus poorly transferred from one protein to another.
A novel extracellular esterase from Bacillus subtilis and its conversion to a monoacylglycerol hydrolase
Authors: Eggert Thorsten , Pencreac‘h Gaëlle , Douchet Isabelle , Verger Robert , Jaeger Karl‐Erich
Published: March 2003

A novel gene lipB, which encodes an extracellular lipolytic enzyme, was identified in the Bacillus subtilis genomic DNA sequence. We have cloned and overexpressed lipB in B. subtilis and Escherichia coli and have also purified the enzyme from a B. subtilis culture supernatant to electrophoretic homogeneity. Four different lipase assays were used to determine its catalytic activity: pH‐stat, spectrophotometry, fluorimetry and the monomolecular film technique. LipB preferentially hydrolysed triacylglycerol‐esters and p‐nitrophenyl‐esters of fatty acids with short chain lengths of ≤ 10 carbon atoms. Triolein, which is a typical substrate for true lipases, was not hydrolysed at all. These results led us to classify LipB as an esterase rather than a lipase. The catalytic triad of LipB consists of residues Ser78, Asp134, and His157 as demonstrated by amino‐acid sequence alignments and site‐directed mutagenesis. The nucleophile Ser78 is located in a lipase‐specific consensus sequence, which is Ala‐X‐Ser‐X‐Gly for most Bacillus lipases. All other bacterial lipases contain a glycine residue instead of the alanine at position‐2 with respect to the catalytic serine. We have investigated the role of this alanine residue by constructing LipB variant A76G, thereby restoring the lipase‐specific consensus motif. When compared with LipB this variant showed a markedly reduced thermostability but an increased stability at pH 5–7. Determination of the specific activities of wild‐type LipB and variant A76G using a monomolecular film of the substrate monoolein revealed an interesting result: the A76G substitution had converted the esterase LipB into a monoacylglycerol hydrolase.
Domain structure of tropomodulin
Authors: Kostyukova Alla , Maeda Kayo , Yamauchi Emiko , Krieger Inna , Maéda Yuichiro
Published: March 2003

The structure of tropomodulin, the unique capping protein for the pointed end (the slow‐growing end) of an actin filament, was studied. An improved Escherichia coli expression system for chicken E‐tropomodulin was established and tropomodulin was prepared, Tmod (N39), in which 15 amino acid residues from the original C‐terminus are deleted at the DNA level. This expression and purification system accidentally co‐produces an 11‐kDa fragment with the original N‐terminus (N11). By applying limited proteolysis to Tmod (N39), a 20‐kDa C‐terminal fragment (C20) was obtained. The limited proteolysis data, as well as the fluorescence spectrometry and CD analyses of Tmod (N39), C20 and N11, revealed that tropomodulin is an α‐helical protein that consists of two distinct domains. The C‐terminal half (20 kDa) is resistant to proteolysis, which suggests that this domain is tightly folded. In contrast, the N‐terminal half is susceptible to proteolysis, indicating that in solution this half is likely to be extended or to form a highly flexible structure. Cross‐linking experiments with glutaraldehyde indicated that Tmod (N39) and N11 can form complexes with tropomyosin, whereas C20 cannot. This confirms the previous report that the site(s) of interaction with tropomyosin resides in the N‐terminal 11‐kDa region of tropomodulin.
Hev b 9, an enolase and a new cross‐reactive allergen from Hevea latex and molds
Authors: Wagner Stefan , Breiteneder Heimo , Simon‐Nobbe Birgit , Susani Markus , Krebitz Monika , Niggemann Bodo , Brehler Randolf , Scheiner Otto , Hoffmann‐Sommergruber Karin
Published: March 2003

Natural rubber latex allergy is an IgE‐mediated disease that is caused by proteins that elute from commercial latex products. A complementary DNA (cDNA) coding for Hev b 9, an enolase (2‐phospho‐ d‐glycerate hydrolyase) and allergen from latex of the rubber tree Hevea brasiliensis, was amplified by PCR. The PCR primers were designed according to conserved regions of enolases from plants. The obtained cDNA amplification product consisted of 1651 bp and encoded a protein of 445 amino‐acid residues with a calculated molecular mass of 47.6 kDa. Sequence comparisons revealed high similarities of the Hevea latex enolase to mold enolases that have been identified as important allergens. In addition, the crucial amino‐acid residues that participate in the formation of the catalytic site and the Mg2+ binding site of enolases were also conserved. Hevea latex enolase was produced as a recombinant protein in Escherichia coli with an N‐terminal hexahistidyl tag, and purified by affinity chromatography. The yield amounted to 110 mg of purified Hev b 9 per litre of bacterial culture. The recombinant allergen bound IgE from latex, as well as mold‐allergic patients, in immunoblot and ELISA experiments. The natural enolase was isolated from Hevea latex by (NH4)2SO4 precipitation and ion exchange chromatography. The natural and the recombinant (r)Hev b 9 showed equivalent enzymatic activity. Patients’ IgE‐antibodies preincubated with rHev b 9 lost their ability to bind to natural (n) Hev b 9, indicating the identity of the B‐cell epitopes on both molecules. Cross‐reactivity with two enolases from Cladosporium herbarum and Alternaria alternata was determined by inhibition of IgE‐binding to these enolases by rHev b 9. Therefore, enolases may represent another class of highly conserved enzymes with allergenic potentials.
Limited heme synthesis in porphobilinogen deaminase‐deficient mice impairs transcriptional activation of specific cytochrome P450 genes by phenobarbital
Authors: Jover Ramiro , Hoffmann Francine , Scheffler‐Koch Valeria , Lindberg Raija L. P.
Published: March 2003

Heme is not only a very important prosthetic group that modulates the structure and activity of heme proteins but also a regulatory molecule that controls metabolic pathways and the biosynthesis of various proteins. However, investigation into heme regulatory effects in higher vertebrates has been hampered by the lack of a suitable animal model. A knockout mouse with targeted disruption of porphobilinogen deaminase, the third enzyme of the heme pathway, has been generated in our laboratory and used in the present study as an in vivo model of heme deficiency to explore diverse heme regulatory properties. In this model with a defined heme disturbance, we observed a superinductive response of δ‐aminolevulinate synthase, the first enzyme in heme synthesis, after phenobarbital treatment. We also found that limited heme is associated with decreased induction of cytochrome P450 by phenobarbital as a consequence of impaired gene transcription. This inhibitory effect is isoenzyme‐specific, being significant for cyp2a5. The activity and mRNA level of this particular cytochrome P450 are significantly lower in the phenobarbital‐induced porphobilinogen deaminase‐deficient mice (55% and 43%, respectively), but its expression can be restored to normal values when exogenous heme is administered. Other heme proteins, namely neuronal nitric oxide synthase and soluble guanylate cyclase, function normally in mice with limited heme. Our results demonstrate that the expression of various heme proteins is differentially regulated in conditions of reduced heme availability. Moreover, our findings emphasize the importance of heme protein function in the genesis of pathophysiological manifestations in acute intermittent porphyria.