bluuebunny/crossref_metadata_2025 huggingface dataset:
Domain structure and stability of human phenylalanine hydroxylase inferred from infrared spectroscopy
Authors: Chehin Rosana , Thorolfsson Matthias , Knappskog Per M , Martinez Aurora , Flatmark Torgeir , Arrondo Jose Luis R , Muga Arturo
Published: July 2002

We have studied the conformation and thermal stability of recombinant human phenylalanine hydroxylase (hPAH) and selected truncated forms, corresponding to distinct functional domains, by infrared spectroscopy. The secondary structure of wild‐type hPAH was estimated to be 48% α‐helix, 28% extended structures, 12% β‐turns and 12% non‐structured conformations. The catalytic C‐terminal domain (residues 112–452) holds most of the regular secondary structure elements, whereas the regulatory N‐terminal domain (residues 2–110) adopts mainly an extended and disordered, flexible conformation. Thermal stability studies of the enzyme forms indicate the existence of interactions between the two domains. Our results also demonstrate that the conformational events involved in the activation of hPAH by its substrate (L‐Phe) are mainly related to changes in the tertiary/quaternary structure. The activating effect of phosphorylation, however, affects the secondary structure of the N‐terminal domain of the protein.
Stimulation of G‐protein coupled receptors in vascular smooth muscle cells induces tyrosine kinase dependent increases in calcium without tyrosine phosphorylation of phospholipase C γ‐1
Authors: Di Salvo Joseph , Raatz Nelson Stephanie
Published: July 2002

It is often believed that increases in intracellular Ca2+ ([Ca2+]i) resulting from stimulation of G‐protein coupled receptors in vascular smooth muscle cells (VSMC) require activation of the β1 isoform of phospholipase C (PLC). However, recent studies showed that rat aortic VSMC do not express PLC β‐1 and that stimulation with angiotensin‐II induces tyrosine kinase dependent increases in [Ca2+]i and tyrosine phosphorylation of PLC γ‐1. Whether this pathway is activated by other vasoactive agents that stimulate G‐protein coupled receptors is unknown. Here, we show that A10 VSMC express PLC β‐2, PLC β‐3, PLC δ‐1, and PLC γ‐1. The cells also expressed Gαq/11. However, neither PLC β‐1 nor PLC β‐4 was detected. Stimulation with angiotensin‐II, vasopressin, serotonin, or endothelin induced tyrosine kinase dependent increases in [Ca2+]i. However, tyrosine phosphorylation of PLC γ‐1 did not occur. In contrast, stimulation with platelet derived growth factor increased [Ca2+]i and tyrosine phosphorylation of PLC γ‐1. The results show that tyrosine phosphorylation of PLC γ‐1 is not required for tyrosine kinase dependent increases in [Ca2+]i resulting from stimulation of diverse G‐protein coupled receptors in VSMC.
Baculovirus‐mediated expression of a scorpion depressant toxin improves the insecticidal efficacy achieved with excitatory toxins
Authors: Gershburg Eduard , Stockholm Daniel , Froy Oren , Rashi Sharon , Gurevitz Michael , Chejanovsky Nor
Published: July 2002

The insecticidal efficacy towards Helicoverpa armigera lepidopteran larvae of recombinant Autographa californica M nucleopolyhedroviruses, expressing depressant and excitatory scorpion anti‐insect selective toxins, was investigated. The ET50 (effective paralysis time 50%) values obtained with the recombinant viruses expressing the depressant toxin, LqhIT2, and the excitatory toxin, LqhIT1, were 59 h and 66 h, respectively, whereas the ET50 value of the wild‐type virus was longer, 87 h post infection. The insecticidal effects obtained when using two distinct temporally regulated viral promoters revealed advantage for the very late p10 promoter over the p35 early promoter. The higher insecticidity of the virus expressing the depressant toxin compared to the excitatory toxin suggests that pharmacokinetic factors and/or promoter efficiency may play a role during infection of insect pest larvae by recombinant baculoviruses.
Adsorption of human lysozyme onto hydroxyapatite
Authors: Aizawa Tomoyasu , Koganesawa Nozomi , Kamakura Akito , Masaki Kazuo , Matsuura Atsushi , Nagadome Hatsumi , Terada Yoshihiro , Kawano Keiichi , Nitta Katsutoshi
Published: July 2002

To elucidate hydroxyapatite‐protein interaction, mutant human lysozymes in which the surface charge was modified by site‐directed mutagenesis were used. Five mutant human lysozymes (K1A, K13A, K33A, R10A, R14A) were expressed in yeast. The chromatographic behavior of these lysozymes was studied with a HPLC hydroxyapatite column. Elution molarities of K1A and R14A mutants were greatly lowered. While Lys‐13 and Arg‐10 are located around Lys‐1 and Arg‐14, K13A and R10A mutants bound onto hydroxyapatite stronger than K1A and R14A mutants. In combination with an X‐ray crystal structure of human lysozyme, it is concluded that the adsorbing site of human lysozyme is at the back of the active site and that Arg‐14, Lys‐1, Arg‐10 and Lys‐13 play important roles in binding.
Rat messenger RNA for the retinal pigment epithelium‐specific protein RPE65 gradually accumulates in two weeks from late embryonic days1
Authors: Manès Gaël , Leducq Régine , Kucharczak Jérôme , Pagès Anne , Schmitt-Bernard Clair Florent , Hamel Christian P
Published: July 2002

The RPE65 protein appears late during the retinal development. To study the basis for this regulation, the rat RPE65 cDNA was sequenced and the mRNA subsequently quantitated at various stages by competitive RT‐PCR. RPE65 mRNA was detected as early as E18 (36 copies/ng of whole eye total RNA). It gradually accumulates up to P12 (27 000 copies/ng) at which point it reaches a steady state level. This increase is interrupted for 3 days (P2–P4) during which the levels of mRNA remain stable. This timing and rate of accumulation parallels that of rat and mouse opsin mRNA and suggests that common factors may control the activation of genes in photoreceptors and retinal pigment epithelium cells.
Diadenosine 5′,5″‐P1,P5‐pentaphosphate harbors the properties of a signaling molecule in the heart
Authors: Jovanovic Aleksandar , Jovanovic Sofija , Mays Dennis C , Lipsky James J , Terzic Andre
Published: July 2002

Dinucleotide polyphosphates (ApnA) have emerged as signaling molecules in rapidly dividing cells. The presence and role of Ap5A in the heart remain unknown. Here, we report that the myocardium contains abundant amounts of diadenosine 5′,5″‐P1,P5‐pentaphosphate (Ap5A), a member of the ApnA family. Ischemia induced 10‐fold decrease in the myocardial concentration of Ap5A. A target of Ap5A action was identified to be the cardiac ATP‐sensitive K+ (KATP) channel, a metabolism‐sensitive ion conductance activated in ischemia. At levels found in hearts prior to ischemia, Ap5A maintained a low probability of KATP channel opening, but at levels found in harts following ischemia, Ap5A allowed a high probability of KATP channel opening. Taken together, the present data suggest that Ap5A harbors the properties of a signaling molecule involved in the cardiac response to metabolic stress.
Cationic uncouplers of oxidative phosphorylation are inducers of mitochondrial permeability transition
Authors: Shinohara Yasuo , Bandou Shidzuko , Kora Shinichi , Kitamura Seiichiro , Inazumi Shuuji , Terada Hiroshi
Published: July 2002

To determine whether cationic uncouplers of oxidative phosphorylation induce permeability transition in mitochondria, the effects of the divalent cationic sulfhydryl cross‐linker copper‐o‐phenanthroline (Cu(OP)2) and the cyanine dye tri‐S‐C4(5) on rat liver mitochondria were examined. Like Ca2+, they accelerated mitochondrial respiration with succinate and induced mitochondrial swelling when inorganic phosphate (Pi) was present in the incubation medium. The acceleration of respiration and swelling were inhibited by the SH‐reagent N‐ethylmaleimide, and by the specific permeability transition inhibitor cyclosporin A (CsA). In addition, these cations, like Ca2+, induced release of ADP entrapped in the mitochondrial matrix space, and the morphological change of mitochondria induced by these cations was essentially the same as that induced by Ca2+. It is concluded that the uncoupling actions of Cu(OP)2 and tri‐S‐C4(5) are due to induction of permeability transition in the inner mitochondrial membrane.
Protein kinase C inhibitors stimulate arachidonic and docosahexaenoic acids release from uterine stromal cells through a Ca2+‐independent pathway
Authors: Birbes Hélène , Pageaux Jean-François , Fayard Jean-Michel , Lagarde Michel , Laugier Christian
Published: July 2002

The mechanisms underlying arachidonic acid (AA) release by uterine stromal (UIII) cells were studied. Stimulation of AA release by calcium ionophore and PMA are inhibited by various PKC inhibitors and by calcium deprivation. These results suggest the involvement of an AA‐specific cPLA2 as the release of docosahexaenoic acid (DHA) from prelabelled cells is much lower than the release of AA. The results also show a more original stimulation of AA and DHA release induced by PKC inhibitors, which is insensitive to calcium deprivation. This stimulation is not due to acyltransferase inhibition, suggesting the participation of a Ca2+‐independent PLA2 (iPLA2). However, iPLA2 activity measured in UIII cells is inhibited by the specific iPLA2 inhibitor, BEL, and is not stimulated by PKC inhibitors, in contrast with the AA and DHA release. It seems therefore that this iPLA2 cannot be involved in this mechanism. The participation of another iPLA2, BEL‐insensitive, is discussed.
Unfolding of bacteriophage P22 tailspike protein: enhanced thermal stability of an N‐terminal fusion mutant
Authors: Carbonell X. , Villaverde A.
Published: July 2002

The tailspike protein (TSP) of bacteriophage P22 is a homotrimeric multifunctional protein responsible for cell attachment and hydrolysis of the Salmonella typhimurium host cell receptor. Despite the folding of TSP involves the formation of thermolabile intermediates, the mature protein is extremely resistant to heat and detergent denaturation. We have analyzed the thermal resistance and unfolding pathway of two mutant, functional TSPs carrying end‐terminal peptide fusions. Whereas the C‐terminal fusion has minor effects on the TSP stability, the presence of a 23‐mer foreign peptide at the N terminus (protein ATSP) results in a significant enhancement of the thermal resistance by retarding the first transition step of the unfolding process. At 65°C and in 2% SDS, the unfolding rate constant for the transition from the native to the unfolding intermediate is 9.3×10−4 s−1 for ATSP versus 1.7×10−3 s−1 for wild‐type TSP. On the other hand, the electrophoretic mobility of ATSP intermediates is greatly affected, proving structural modifications induced by the fused peptide. These results suggest a critical participation of the N‐terminal domain in the unfolding kinetic barriers generated during the TSP denaturation pathway.
The effects of SNAP/SNARE complexes on the ATPase of NSF
Authors: Matveeva Elena , Whiteheart Sidney W.
Published: July 2002

The ATPase of the N‐ethylmaleimide sensitive factor (NSF) appears to be central to the events that culminate in vesicle‐target membrane fusion. Complexes containing different combinations of NSF, α‐SNAP, Vamp‐2 (synaptobrevin 2), syntaxin 1, and SNAP‐25 were reconstituted and then tested for their effect on the ATPase of NSF. While NSF interacts with all α‐SNAP‐containing complexes, only the α‐SNAP/t‐SNARE complex significantly stimulated ATPase activity. This stimulation was dependent on increasing SNAP/t‐SNARE complex and was saturable. The apparent stimulation of ATPase activity is due to a 10‐fold increase in initial hydrolysis rate. Complex containing both v‐ and t‐SNAREs bound significantly more α‐SNAP but did not stimulate the ATPase of NSF.
Thermoregulatory uncoupling in heart muscle mitochondria: involvement of the ATP/ADP antiporter and uncoupling protein
Authors: Simonyan Ruben A , Skulachev Vladimir P
Published: July 2002

Possible involvement of the ATP/ADP antiporter and uncoupling protein (UCP) in thermoregulatory uncoupling of oxidative phosphorylation in heart muscle has been studied. To this end, effects of carboxyatractylate (cAtr) and GDP, specific inhibitors of the antiporter and UCP, on the membrane potential of the oligomycin‐treated mitochondria from cold‐exposed (6°C, 48 h) and control rats have been measured. It is found that cAtr increases the membrane potential level in both cold‐exposed and non‐exposed groups, the effect being strongly enhanced by cooling. As for GDP, it is effective only in mitochondria from the cold‐exposed rats. In these mitochondria, the coupling effect of GDP is smaller than that of cAtr. CDP, which does not interact with UCP, is without any influence on membrane potential. The cold exposure is found to increase the uncoupling efficiency of added natural (palmitate) or artificial (SF6847) uncouplers, the increase being cAtr‐ and GDP‐sensitive in the case of palmitate. The fatty acid‐free bovine serum albumin enhances ΔΨ in both cold‐exposed and control groups, the effect being much larger in the former case. It is concluded that in heart muscle mitochondria the ATP/ADP antiporter is responsible for the ‘mild uncoupling’ under normal conditions and for major portion of the thermoregulatory uncoupling in the cold whereas the rest of thermoregulatory uncoupling is served by UCP (presumably by UCP2 since the UCP2 mRNA level is shown to strongly increase in rat heart muscle under the cold exposure conditions used).
In vivo phosphorylation of poly(ADP‐ribose) polymerase is independent of its activation
Authors: Ariumi Yasuo , Ueda Kunihiro , Masutani Mitsuko , Copeland Terry D , Noda Makoto , Hatanaka Masakazu , Shimotohno Kunitada
Published: July 2002

Poly(ADP‐ribose) polymerase (PARP) is a nuclear enzyme, which is activated by DNA strand breaks. Although PARP is known to be cleaved by the cysteine protease, caspase‐3/CPP32, during apoptosis, signal cascade which regulates the PARP activity has not been fully understood. In this study, we investigated post‐translational modification of PARP. We found that PARP was phosphorylated by a serine kinase in vivo. PARP was activated temporarily and extensive auto‐modification occurred on PARP, possibly by the fragmented DNA during apoptosis induced by etoposide in Jurkat cells. However, the phosphorylation level was not changed for up to 6 h, after PARP cleavage began in apoptosis by the treatment with etoposide. Furthermore, we showed the presence of a PARP‐associated kinase in nuclear extracts of the HTLV‐I infected T‐cell lines but not in uninfected T‐cell lines, whereas this kinase did not inhibit the PARP activity even in the presence of ATP. Taken together, in vivo phosphorylation of PARP might be independent of the activation or cleavage of PARP.
β‐Carotene with vitamins E and C offers synergistic cell protection against NOx
Authors: Böhm F. , Edge R. , McGarvey D.J. , Truscott T.G.
Published: July 2002

The peroxynitrite anion and the nitrogen dioxide (radical) are important toxic species which can arise in vivo from nitric oxide. Both in vivo and in vitro cell protection is demonstrated for β‐carotene in the presence of vitamin E and vitamin C. A synergistic protection is observed compared to the individual anti‐oxidants and this is explained in terms of an electron transfer reaction in which the β‐carotene radical is repaired by vitamin C.
Promoter activation and following induction of the p21/WAF1 gene by flavone is involved in G1 phase arrest in A549 lung adenocarcinoma cells
Authors: Bai Fulu , Matsui Takayoshi , Ohtani-Fujita Naoko , Matsukawa Yoshizumi , Ding Yi , Sakai Toshiyuki
Published: July 2002

Flavonoids are present in many plants including edible fruits and vegetables. Recently, many of the biological activities of flavonoids have been elucidated. Flavone is a well known flavonoid, and many of its derivatives have been shown to have anti‐proliferative effects on several cancer cells. We report here that flavone can effectively inhibit the cell growth of human lung adenocarcinoma A549 cells in a dose‐dependent manner, and 100 μM flavone causes cell cycle arrest at the G1 phase. As a mechanism underlying the cell cycle arrest, flavone markedly increases the mRNA and protein levels of a universal inhibitor of cyclin‐dependent kinase, p21/WAF1, and inhibits phosphorylation of retinoblastoma (RB) protein. Although A549 cells possess wild‐type p53, flavone does not induce the p53 protein, suggesting that p21/WAF1 induction is p53‐independent. In addition, 100 μM flavone significantly increases the promoter activity of the p21/WAF1 gene by 5‐fold. These results suggest that the G1 phase arrest by flavone is due to p53‐independent transcriptional induction of the p21/WAF1 gene and the subsequent dephosphorylation of RB protein.
C‐terminal domains of human translation termination factors eRF1 and eRF3 mediate their in vivo interaction
Authors: Merkulova Tatyana I. , Frolova Lyudmila Y. , Lazar Monique , Camonis Jacques , Kisselev Lev L.
Published: July 2002

At the termination step of protein synthesis, hydrolysis of the peptidyl‐tRNA is jointly catalysed at the ribosome by the termination codon and the polypeptide release factor (eRF1 in eukaryotes). eRF1 forms in vivo and in vitro a stable complex with release factor eRF3, an eRF1‐dependent and ribosome‐dependent GTPase. The role of the eRF1⋅eRF3 complex in translation remains unclear. We have undertaken a systematic analysis of the interactions between the human eRF1 and eRF3 employing a yeast two‐hybrid assay. We show that the N‐terminal parts of eRF1 (positions 1–280) and of eRF3 (positions 1–477) are either not involved or non‐essential for binding. Two regions in each factor are critical for mutual binding: positions 478–530 and 628–637 of eRF3 and positions 281–305 and 411–415 of eRF1. The GTP binding domain of eRF3 is not involved in complex formation with eRF1. The GILRY pentamer (positions 411–415) conserved in eukaryotes and archaebacteria is critical for eRF1's ability to stimulate eRF3 GTPase. The human eRF1 lacking 22 C‐terminal amino acids remains active as a release factor and promotes an eRF3 GTPase activity whereas C‐terminally truncated eRF3 is inactive as a GTPase.
AT2 receptor stimulation induces generation of ceramides in PC12W cells
Authors: Gallinat Stefan , Busche Silke , Schütze Stefan , Krönke Martin , Unger Thomas
Published: July 2002

The angiotensin AT2 receptor has been implicated in both regeneration and apoptosis. To further investigate the molecular mechanisms leading to AT2 receptor‐induced programmed cell death in PC12W cells we studied the effects of angiotensin II (ANG II) on ceramide levels by HPTLC analysis. We could demonstrate that ANG II time‐ (1–10 h) and dose‐dependently (10−8–5×10−6 M) increased ceramide levels by maximally 175% but did not affect sphingomyelin degradation. The ANG II effects were mediated by AT2 receptors since they were completely abolished by co‐incubation with the AT2 receptor antagonist, PD123177 (10−5 M), but not by the AT1 receptor antagonist, losartan (10−5 M). These data suggest a novel signal transduction pathway to the AT2 receptor leading to apoptosis in neuronal cells.
Physiological production of singlet molecular oxygen in the myeloperoxidase‐H2O2‐chloride system
Authors: Kiryu Chika , Makiuchi Masao , Miyazaki Junji , Fujinaga Toru , Kakinuma Katsuko
Published: July 2002

The putative role of singlet oxygen (1O2) in the respiratory burst of neutrophils has remained elusive due to the lack of reliable means to study its quantitative production. To measure 1O2 directly from biological or chemical reactions in the near infrared region, we have developed a highly sensitive detection system which employs two InGaAs/InP pin photodiodes incorporated with a dual charge integrating amplifier circuit. Using this detection system, we detected light emission derived from a myeloperoxidase (MPO)‐mediated reaction in physiological conditions: pH 7.4, 1–30 nM MPO, 10–100 μM H2O2 and 100–130 mM Cl− in place of Br− without the use of deuterium oxide. The MPO‐H2O2‐Cl− system exhibited a single emission peak at 1.27 μm with a spectral distribution identical to that of delta singlet oxygen. Our results suggest physiological production of 1O2 in the MPO‐H2O2‐Cl− system at an intravacuolar neutral pH. The MPO‐mediated generation of 1O2, which may have an important role in host defense mechanisms, is discussed in connection with previous results.
The lipopolysaccharide‐binding protein participating in hemocyte nodule formation in the silkworm Bombyx mori is a novel member of the C‐type lectin superfamily with two different tandem carbohydrate‐recognition domains1
Authors: Koizumi Nobuo , Imamura Morikazu , Kadotani Tomoyuki , Yaoi Katsuro , Iwahana Hidenori , Sato Ryoichi
Published: July 2002

We recently isolated and characterized the lipopolysaccharide (LPS)‐binding protein, BmLBP, from the larval hemolymph of the silkworm Bombyx mori. BmLBP is a pattern recognition molecule that recognizes the lipid A portion of LPS and participates in a cellular defense reaction. This paper describes the cDNA cloning of BmLBP. The deduced amino acid sequence of BmLBP revealed that BmLBP is a novel member of the C‐type lectin superfamily with a unique structural feature that consists of two different carbohydrate‐recognition domains in tandem, a short and a long form.
Phe161 and Arg166 variants of p‐hydroxybenzoate hydroxylase
Authors: Eppink Michel H.M. , Bunthof Christine , Schreuder Herman A. , van Berkel Willem J.H.
Published: July 2002

Phe161 and Arg166 of p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens belong to a newly discovered sequence motif in flavoprotein hydroxylases with a putative dual function in FAD and NADPH binding [1]. To study their role in more detail, Phe161 and Arg166 were selectively changed by site‐directed mutagenesis. F161A and F161G are catalytically competent enzymes having a rather poor affinity for NADPH. The catalytic properties of R166K are similar to those of the native enzyme. R166S and R166E show impaired NADPH binding and R166E has lost the ability to bind FAD. The crystal structure of substrate complexed F161A at 2.2 Å is indistinguishable from the native enzyme, except for small changes at the site of mutation. The crystal structure of substrate complexed R166S at 2.0 Å revealed that Arg166 is important for providing an intimate contact between the FAD binding domain and a long excursion of the substrate binding domain. It is proposed that this interaction is essential for structural stability and for the recognition of the pyrophosphate moiety of NADPH.
Rho‐kinase in human neutrophils: a role in signalling for myosin light chain phosphorylation and cell migration
Authors: Niggli Verena
Published: July 2002

The role of a Rho‐associated coiled‐coil forming kinase in migration of neutrophils has been investigated. Rho‐associated coiled‐coil forming kinase I was expressed in human neutrophils. Chemotactic peptide led to a Rho‐associated coiled‐coil forming kinase‐dependent increase in phosphorylation of myosin light chain. This was determined with the help of an antibody directed against serine 19‐phosphorylated myosin light chain and an inhibitor of Rho‐associated coiled‐coil forming kinase (Y‐27632). Y‐27632 suppressed myosin light chain phosphorylation and chemotactic peptide‐induced development of cell polarity and locomotion with similar potency (ED50 0.5–1.1 μM). The data strongly suggest that a Rho‐associated coiled‐coil forming kinase isoform, activated in human neutrophils exposed to chemotactic peptide, is important for motile functions of these cells.
Ascorbate prevents prooxidant effects of urate in oxidation of human low density lipoprotein
Authors: Abuja Peter M
Published: July 2002

Uric acid and ascorbic acid are important low molecular weight antioxidants in plasma. Their interactions and combined effect on Cu2+‐catalysed oxidation of human low density lipoprotein were studied in vitro. It was found that uric acid alone becomes strongly prooxidant whenever it is added to low density lipoprotein shortly after the start of oxidation (conditional prooxidant). Ascorbic acid, which is present in human plasma at much lower concentrations (20–60 μM) than urate (300–400 μM), is in itself not a conditional prooxidant. Moreover, ascorbate prevents prooxidant effects of urate, when added to oxidising low density lipoprotein simultaneously with urate, even at a 60‐fold molar excess of urate over ascorbate. Ascorbate appears to have the same anti‐prooxidant effect with other aqueous reductants, which, besides their antioxidant properties, were reported to be conditionally prooxidant. Such interactions between ascorbate and urate may be important in preventing oxidative modification of lipoproteins in the circulation and in other biological fluids.
Molecular cloning of rat GADD45γ, gene induction and its role during neuronal cell death
Authors: Kojima Satoshi , Mayumi-Matsuda Keiko , Suzuki Harukazu , Sakata Tsuneaki
Published: July 2002

To study the molecular mechanism of neuronal cell death, we carried out the screening of genes which were induced during the neuronal cell death of neuronal PC12. We cloned the cDNA of rat GADD45γ, the third member of the GADD45 family. Induction of GADD45γ mRNA was observed in the neuronal cell death caused by depletion of neurotrophic factor and Ca2+ ionophore treatment. Overexpression of GADD45γ in superior cervical ganglion neurons caused cell death. These results suggest that GADD45γ plays an important role in neuronal cell death.
Prevention of αIIbβ3 activation by non‐steroidal antiinflammatory drugs
Authors: Domı́nguez-Jiménez Carmen , Dı́az-González Federico , González-Álvaro Isidoro , Cesar Jesús M. , Sánchez-Madrid Francisco
Published: July 2002

We have studied the effect of non‐steroidal antiinflammatory drugs (NSAIDs) on αIIbβ3 integrin activation and platelet aggregation. NSAIDs such as meloxicam, piroxicam, indomethacin and aspirin, but not aceclofenac or diclofenac interfered with the activation state of αIIbβ3. NSAIDs that inhibited αIIbβ3 activation were also able both to partially inhibit platelet primary aggregation and to accelerate platelet deaggregation. These effects of NSAIDs were not dependent on cyclooxygenase inhibition. The results obtained indicate that some NSAIDs exert a specific action on αIIbβ3 activation, and provide an additional mechanism that accounts for their beneficial effects in diseases in which platelet activation is involved.
Biliary excretion of copper in LEC rat after introduction of copper transporting P‐type ATPase, ATP7B
Authors: Terada Kunihiko , Aiba Namiko , Yang Xiao-Li , Iida Masatake , Nakai Michio , Miura Naoyuki , Sugiyama Toshihiro
Published: July 2002

Wilson's disease, an autosomal recessive disorder, is characterized by the excessive accumulation of hepatic copper that results from reduced biliary copper excretion and disturbed incorporation of copper into ceruloplasmin. The ATP7B gene, responsible for the disease, encodes a copper transporting P‐type ATPase. We previously demonstrated the involvement of ATP7B in hepatic copper secretion into plasma after the introduction of ATP7B into the Long‐Evans Cinnamon (LEC) rat, a rodent model of Wilson's disease. In this study we found the increased copper contents of the hepatic lysosomal fractions and bile in the LEC rats after ATP7B introduction, indicating the participation of ATP7B in the biliary excretory pathway for copper.
Biological activities of C‐terminal 15‐residue synthetic fragment of melittin: design of an analog with improved antibacterial activity
Authors: Subbalakshmi C. , Nagaraj R. , Sitaram N.
Published: July 2002

Melittin, the 26‐residue predominant toxic peptide from bee venom, exhibits potent antibacterial activity in addition to its hemolytic activity. The synthetic peptide of 15 residues corresponding to its C‐terminal end (MCF), which encompasses its most amphiphilic segment, is now being shown to possess antibacterial activity about 5–7 times less compared to that of melittin. MCF, however, is 300 times less hemolytic. An analog of MCF, MCFA, in which two cationic residues have been trans‐positioned to the N‐terminal region from the C‐terminal region, exhibits antibacterial activity comparable to that of melittin, but is only marginally more hemolytic than MCF. The biophysical properties of the peptides, like folding and aggregation, correlate well with their biological properties.
On the mechanism of hepatic transendothelial passage of large liposomes
Authors: Romero Eder L. , Morilla Maria-José , Regts Joke , Koning Gerben A. , Scherphof Gerrit L.
Published: July 2002

Liposomes of 400 nm in diameter can cross the 100‐nm fenestrations in the endothelium of the hepatic sinusoid, provided they contain phosphatidylserine (PS) but not phosphatidylglycerol (PG) [Daemen et al. (1997) Hepatology 26, 416]. We present evidence indicating that (i) the PS effect does not involve a pharmacological action of this lipid on the size of the fenestrations, (ii) fluid‐type but not solid‐type PS liposomes have access to the hepatocytes and (iii) the lack of uptake of PG liposomes by hepatocytes is not due to a lack of affinity of the hepatocytes for PG surfaces. We conclude that the mechanism responsible for the uptake of large PS‐containing liposomes by hepatocytes in vivo involves a mechanical deformation of these liposomes during their passage across the endothelial fenestrations.
Mutations of the mitochondrially encoded ATPase 6 gene modeled in the ATP synthase of Escherichia coli
Authors: Ogilvie Isla , Capaldi Roderick A.
Published: July 2002

Defects of respiratory chain protein complexes and the ATP synthase are becoming increasingly implicated in human disease. Recently, mutations in the ATPase 6 gene have been shown to cause several different neurological disorders. The product of this gene is homologous to the a subunit of the ATP synthase of Escherichia coli. Here, mutations equivalent to those described in humans have been introduced into the a subunit of E. coli by site‐directed mutagenesis, and the effects of these mutations on the ATPase activity, ATP synthesis and ability of the enzyme to pump protons studied in detail. The effects of the mutations varied considerably. The mutation L262P (9185 T‐C equivalent) caused a 70% loss of ATP synthesis activity, reduced DCCD sensitivity, and lowered proton pumping activity. The L207P (8993 T‐C equivalent) reduced ATP synthesis by 50%, affected DCCD sensitivity, while proton pumping was only marginally affected when measured by the standard AMCA quenching assay. The other mutations studied affected the functioning of the ATP synthase much less. The results confirm that modeling of these point mutations in the E. coli enzyme is a useful approach to determining how alterations in the ATPase 6 gene affect enzyme function and, therefore, how a pathogenic effect can be exerted.
Structural analysis of a plant sucrose carrier using monoclonal antibodies and bacteriophage lambda surface display
Authors: Stolz Jürgen , Ludwig Andreas , Stadler Ruth , Biesgen Christian , Hagemann Klaus , Sauer Norbert
Published: July 2002

Monoclonal antibodies were raised and selected against recombinant Plantago major PmSUC2 sucrose carrier protein. Epitopes of two monoclonal antibodies (PS2‐1A2 and PS2‐4D4) were mapped using N‐terminally truncated PmSUC2 proteins and a lambda library displaying random PmSUC2 peptides. PS2‐1A2 recognizes an octapeptide close to the N‐terminus of PmSUC2, PS2‐4D4 binds to a decapeptide at the very C‐terminus. Analyses of antibody binding to yeast protoplasts with functionally active, tagged PmSUC2 protein revealed that both epitopes are located in cytoplasmic domains of PmSUC2. These results support a model for plant sucrose transporters containing 12 transmembrane helices with the N‐terminus and the C‐terminus on the cytoplasmic side of the plasma membrane.
Amyloid‐like aggregates of a plant protein: a case of a sweet‐tasting protein, monellin
Authors: Konno Takashi , Murata Kazuyoshi , Nagayama Kuniaki
Published: July 2002

We report here a novel case of amyloid‐like aggregation of a plant protein. A sweet‐tasting protein, monellin, experiences an irreversible heat denaturation at pH 2.5 and 85°C. Addition of 100 mM NaCl couples this process with protein aggregation. The aggregates were structured as regular fibers with ∼10 nm width and capable of binding to Congo red, similarly to well‐known amyloid fibrils. The amyloid‐like aggregation process was also successfully monitored with a calorimetric method. This work supports the universality of the amyloid‐like aggregation, not restricted to some special categories of protein.
Selection of ganglioside GM1‐binding peptides by using a phage library
Authors: Matsubara Teruhiko , Ishikawa Dai , Taki Takao , Okahata Yoshio , Sato Toshinori
Published: July 2002

Ganglioside Galβ1→3GalNAcβ1→4(NeuAcα2→3)Galβ1→4Glcβ1→1'Cer (GM1)‐binding peptides were obtained from a phage‐displayed pentadecapeptide library by an affinity selection. The selection processes were in situ‐monitored by a quartz‐crystal microbalance method, on which a ganglioside GM1 monolayer was transferred. After five rounds of biopanning, the DNA sequencing of 18 selected phages showed that only three individual clones were selected. The peptide sequences of the random region were found to be DFRRLPGAFWQLRQP, GWWYKGRARPVSAVA and VWRLLAPPFSNRLLP. Binding constants of these phage clones to the GM1 monolayer were 1010 M−1. Three synthetic pentadecapeptides inhibited the binding of cholera toxin B subunit to the GM1 monolayer with an IC
50 of 24, 13 and 1.0 μM, respectively. These peptides will be useful for searching functional roles of ganglioside GM1.
TGF‐β inhibits lipopolysaccharide‐stimulated activity of c‐Jun N‐terminal kinase in mouse macrophages
Authors: Imai Kenichi , Takeshita Akira , Hanazawa Shigemasa
Published: July 2002

Transforming growth factor‐β (TGF‐β) is a potent anti‐inflammatory cytokine. Although this cytokine inhibits lipopolysaccharide (LPS)‐mediated septic shock, the molecular mechanism of TGF‐β is not well known. Since recent studies showed that c‐Jun N‐terminal kinase (JNK), one of the mitogen‐activated protein kinases, plays an important role in LPS signalling, we focused here on the inhibitory action of TGF‐β1 on LPS‐stimulated JNK activity in mouse macrophages. TGF‐β1 inhibited LPS‐stimulation of phosphorylated JNK1 and JNK2 and consequently of JNK activity in the cells. This JNK activity resulted in a decreased level of phosphorylated c‐Jun protein. Using Western blotting, we also observed TGF‐β1 inhibition of newly synthesized c‐Jun protein in LPS‐stimulated cells. These results demonstrate that TGF‐β1 inhibits LPS‐stimulated JNK activity in mouse macrophages. Also, our present study suggests a possible inhibitory mechanism of TGF‐β in signalling of LPS‐induced inflammatory responses.
Characterization of GFR, a novel guanine nucleotide exchange factor for Rap1
Authors: Ichiba Tamotsu , Hoshi Yasutaka , Eto Yoshikatsu , Tajima Naoko , Kuraishi Yasunobu
Published: July 2002

Three groups of Rap1‐specific guanine nucleotide exchange factors including C3G, CalDAG‐GEFI, and Epac/cAMP‐GEFI/II have been identified to date. In the present study, we report a new Rap1 guanine nucleotide exchange factor which we have named GFR (uanine nucleotide exchange actor for ap1). GFR shows close sequence similarity to EPAC/cAMP‐GEFI/II although GFR lacks a cAMP binding domain and contains a nuclear localization signal. We demonstrated that GFR can activate Rap1 but not H‐Ras in 293T cells and that the cdc25 domain of GFR is required for the activation of Rap1. Northern blot analysis suggested that GFR mRNA is strongly expressed in the brain. In transfected HeLa cells, GFR has been found to be localized in the nuclei.
Detergent‐free membrane protein crystallization
Authors: Nollert Peter , Royant Antoine , Pebay-Peyroula Eva , Landau Ehud M.
Published: July 2002

A comprehensive understanding of structure‐function relationships of proteins requires their structures to be elucidated to high resolution. With most membrane proteins this has not been accomplished so far, mainly because of their notoriously poor crystallizability. Here we present a completely detergent‐free procedure for the incorporation of a native purple membrane into a monoolein‐based lipidic cubic phase, and subsequent crystallization of three‐dimensional bacteriorhodopsin crystals therein. These crystals exhibit comparable X‐ray diffraction quality and mosaicity, and identical crystal habit and space group to those of bacteriorhodopsin crystals that are grown from detergent‐solubilized protein in cubic phase.
Time‐resolved generation of a membrane potential by ba3 cytochrome c oxidase from Thermus thermophilus
Authors: Siletskiy S. , Soulimane T. , Azarkina N. , Vygodina T.V. , Buse G. , Kaulen A. , Konstantinov A.
Published: July 2002

ba
3‐type cytochrome c oxidase purified from the thermophilic bacterium Thermus thermophilus has been reconstituted in phospholipid vesicles and laser flash‐induced generation of a membrane potential by the enzyme has been studied in a μs/ms time scale with Ru(II)‐tris‐bipyridyl complex (RuBpy) as a photoreductant. Flash‐induced single electron reduction of the aerobically oxidized ba
3 by RuBpy results in two phases of membrane potential generation by the enzyme with τ values of about 20 and 300 μs at pH 8 and 23°C. Spectrophotometric experiments show that oxidized ba
3 reacts very poorly with hydrogen peroxide or any of the other exogenous heme iron ligands studied like cyanide, sulfide and azide. At the same time, photoreduction of the enzyme by RuBpy triggers the electrogenic reaction with H2O2 with a second order rate constant of ∼2×103 M−1 s−1. The data indicate that single electron reduction of ba
3 oxidase opens the binuclear center of the enzyme for exogenous ligands. The fractional contribution of the protonic electrogenic phases induced by peroxide in cytochrome ba
3 is much less than in bovine oxidase, pointing to a possibility of a different electrogenic mechanism of the ba
3 oxidase as compared to the oxidases of the aa
3‐type.
The cadherin‐like protein is essential to specificity determination and cytotoxic action of the Bacillus thuringiensis insecticidal CryIAa toxin
Authors: Nagamatsu Yasunori , Koike Takashi , Sasaki Kazuhiro , Yoshimoto Akihiro , Furukawa Yasuo
Published: July 2002

The Bacillus thuringiensis CryIAa toxin binds a cadherin‐like protein (BtR175) on the brush‐border membranes of the Bombyx mori midgut columnar cells, which are the targets. By introducing the BtR175 gene with a baculovirus, Spodoptera frugiperda Sf9 cells expressed BtR175 protein on the cell membrane and became susceptible to the CryIAa toxin. The toxin bound the cadherin repeat adjacent to the membrane and made a pore that passed inorganic ions, causing the cell to swell and burst. This was not observed with a BtR175 variant lacking the toxin‐binding site. This in vitro experiment mimicked the specific insecticidal action of the toxin in vivo well.
One‐step purification of the NADH dehydrogenase fragment of the Escherichia coli complex I by means of Strep‐tag affinity chromatography
Authors: Bungert Stefanie , Krafft Bianca , Schlesinger Ramona , Friedrich Thorsten
Published: July 2002

The proton‐pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first energy‐transducing complex of many respiratory chains. Complex I of Escherichia coli can be split into three fragments. One of these fragments, the soluble NADH dehydrogenase fragment, represents the electron input part of complex I. It comprises the subunits NuoE, F and G and harbors one flavin mononucleotide and up to six iron‐sulfur clusters. Here, we report the one‐step purification of this fragment by means of affinity chromatography on StrepTactin. This was achieved by fusing the Strep‐tag II peptide to the C‐terminus of NuoF or NuoG. Fusion of this peptide to the N‐terminus of either NuoE or NuoF disturbed the assembly of the NADH dehydrogenase fragment.
Identification and characterisation of novel polymorphisms in the CYP2A locus: implications for nicotine metabolism
Authors: Oscarson Mikael , McLellan Roman A. , Gullstén Harriet , Agúndez José A.G. , Benı́tez Julio , Rautio Arja , Raunio Hannu , Pelkonen Olavi , Ingelman-Sundberg Magnus
Published: July 2002

The polymorphic human cytochrome P450 2A6 (CYP2A6) metabolises a number of drugs, activates a variety of precarcinogens and constitutes the major nicotine C‐oxidase. A relationship between CYP2A6 genotype and smoking habits, as well as incidence of lung cancer, has been proposed. Two defective alleles have hitherto been identified, one of which is very common in Asian populations. Among Caucasians, an additional defective and frequently distributed allele (CYP2A6*3) has been suggested to play a protective role against nicotine addiction and cigarette consumption. Here, we have re‐evaluated the genotyping method used for the CYP2A6*3 allele and found that a gene conversion in the 3′ flanking region of 30–40% of CYP2A6*1 alleles results in genotype misclassification. In fact, no true CYP2A6*3 alleles were found among 100 Spaniards and 96 Chinese subjects. In one Spanish poor metaboliser of the CYP2A6 probe drug coumarin, we found two novel defective alleles. One, CYP2A6*5, encoded an unstable enzyme having a G479L substitution and the other was found to carry a novel type of CYP2A6 gene deletion (CYP2A6*4D). The results imply the presence of numerous defective as well as active CYP2A6 alleles as a consequence of CYP2A6/CYP2A7 gene conversion events. We conclude that molecular epidemiological studies concerning CYP2A6 require validated genotyping methods for accurate detection of all known defective CYP2A6 alleles.
NMR approaches for monitoring domain orientations in calcium‐binding proteins in solution using partial replacement of Ca2+ by Tb3+
Authors: Biekofsky Rodolfo R. , Muskett Frederick W. , Schmidt Jürgen M. , Martin Stephen R. , Browne J.Peter , Bayley Peter M. , Feeney James
Published: July 2002

This work shows that the partial replacement of diamagnetic Ca2+ by paramagnetic Tb3+ in Ca2+/calmodulin systems in solution allows the measurement of interdomain NMR pseudocontact shifts and leads to magnetic alignment of the molecule such that significant residual dipolar couplings can be measured. Both these parameters can be used to provide structural information. Species in which Tb3+ ions are bound to only one domain of calmodulin (the N‐domain) and Ca2+ ions to the other (the C‐domain) provide convenient systems for measuring these parameters. The nuclei in the C‐domain experience the local magnetic field induced by the paramagnetic Tb3+ ions bound to the other domain at distances of over 40 Å from the Tb3+ ion, shifting the resonances for these nuclei. In addition, the Tb3+ ions bound to the N‐domain of calmodulin greatly enhance the magnetic susceptibility anisotropy of the molecule so that a certain degree of alignment is produced due to interaction with the external magnetic field. In this way, dipolar couplings between nuclear spins are not averaged to zero due to solution molecular tumbling and yield dipolar coupling contributions to, for example, the one‐bond 15N‐1H splittings of up to 17 Hz in magnitude. The degree of alignment of the C‐domain will also depend on the degree of orientational freedom of this domain with respect to the N‐domain containing the Tb3+ ions. Pseudocontact shifts for NH groups and 1H‐15N residual dipolar couplings for the directly bonded atoms have been measured for calmodulin itself, where the domains have orientational freedom, and for the complex of calmodulin with a target peptide from skeletal muscle myosin light chain kinase, where the domains have fixed orientations with respect to each other. The simultaneous measurements of these parameters for systems with domains in fixed orientations show great potential for the determination of the relative orientation of the domains.
The plasmid F OmpP protease, a homologue of OmpT, as a potential obstacle to E. coli‐based protein production
Authors: Matsuo Ei-ichi , Sampei Gen-ichi , Mizobuchi Kiyoshi , Ito Koreaki
Published: July 2002

OmpT, an outer membrane‐localized protease of Escherichia coli, cleaves a number of exogenous and endogenous proteins during their purification. SecY, an endogenous membrane protein, is a target of this artificial proteolysis in vitro. Here we report that SecY cleavage occurs even in cell extracts from ompT‐disrupted cells, if they carry an F plasmid derivative. A gene, ompP, on the F plasmid was shown to be responsible for this proteolysis. These results indicate that the absence of an F‐like plasmid should be checked when choosing a host strain for E. coli‐based protein production.
Analysis of the promoter of the MUC1 gene overexpressed in breast cancer
Authors: Zaretsky Joseph Z. , Sarid Ronit , Aylon Yael , Mittelman Leonid A. , Wreschner Daniel H. , Keydar Iafa
Published: July 2002

The MUC1 gene encodes a mucin glycoprotein and is overexpressed in breast cancer. Knowledge of the mechanisms leading to MUC1 overexpression may help in the development of molecular approaches for breast cancer therapy. In order to study the regulation of the MUC1 gene transcription, we analyzed functional activities of various deletion mutants of the MUC1 promoter. We established that transcriptional cis‐elements present in the SacI/XmnI fragment of the promoter are competent and sufficient for expression of, at least, tandem repeats containing isoform(s) of the MUC1 protein. CAT transfection analysis showed that both the 3′ and 5′ regions of the SacI/XmnI fragment possess transcription activities. Promoter activities associated with the SacI/XmnI fragment were confirmed by a RNase protection assay, which demonstrated multiple transcription start sites (TSSs) in the MUC1 gene transcribed in epithelial T47D cells. We show that treatment of the T47D cells with TGFβ1 leads to activation of additional TSSs in the MUC1 gene. The roles of the structural and functional properties of the MUC1 promoter in MUC1 gene transcription are discussed.
Evidence for glucose and sorbitol‐induced nuclear export of glucokinase regulatory protein in hepatocytes
Authors: Mukhtar Mohammed , Stubbs Mark , Agius Loranne
Published: July 2002

Glucokinase is rapidly exported from the nucleus of hepatocytes in response to a rise in glucose or fructose 1‐P. We demonstrate using confocal microscopy and quantitative imaging that in contrast to previous findings, the regulatory protein of glucokinase (GKRP) also translocates from the nucleus during substrate‐induced translocation of glucokinase. However, the fractional decrease in nuclear GKRP is smaller than for glucokinase and is determined by the metabolic state and not by the distribution of glucokinase. Translocation of glucokinase and GKRP is not inhibited by leptomycin B, an inhibitor of exportin‐1 function. These findings highlight the importance of quantitative imaging for determining nuclear export of proteins and suggest that GKRP may have a role in nuclear export or import of glucokinase.
Bacillus thuringiensis Cry1Aa toxin‐binding region of Bombyx mori aminopeptidase N
Authors: Yaoi Katsuro , Nakanishi Kazuko , Kadotani Tomoyuki , Imamura Morikazu , Koizumi Nobuo , Iwahana Hidenori , Sato Ryoichi
Published: July 2002

The Bacillus thuringiensis Cry1Aa toxin‐binding region of Bombyx mori aminopeptidase N (APN) was analyzed, to better understand the molecular mechanism of susceptibility to the toxin and the development of resistance in insects. APN was digested with lysylendopeptidase and the ability of the resulting fragments to bind to Cry1Aa and 1Ac toxins was examined. The binding abilities of the two toxins to these fragments were different. The Cry1Aa toxin bound to the fragment containing 40‐Asp to 313‐Lys, suggesting that the Cry1Aa toxin‐binding site is located in the region between 40‐Asp and 313‐Lys, while Cry1Ac toxin bound exclusively to mature APN. Next, recombinant APN of various lengths was expressed in Escherichia coli cells and its ability to bind to Cry1Aa toxin was examined. The results localized the Cry1Aa toxin binding to the region between 135‐Ile and 198‐Pro.