bluuebunny/crossref_metadata_2025 huggingface dataset:
Frequency dependence of Q in Earth's upper mantle inferred from continuous spectra of body waves
Authors: Shito Azusa , Karato Shun‐ichiro , Park Jeffrey
Published: June 2004

The frequency dependence of Q in Earth's upper mantle is investigated using the continuous P wave spectra over 0.08 < f < 8.0 Hz. Regional waveform data from 23 events in the Pacific slab recorded on broadband stations in western Japan were used to create spectral ratio estimates of P waves which sample a subducting slab and mantle wedge. We assumed the power‐law model Q(f) = Q0(f/f0)α and applied a grid search to determine the best fit α and Q0. The inferred frequency dependence (0.2 < α < 0.4) in most regions is consistent with laboratory data on solid olivine but not on partially molten olivine‐rich rocks except those data for deep events. Close agreement between seismological and laboratory observations on frequency dependence of Q suggests that physical mechanisms of seismic wave attenuation are common and therefore laboratory data can be used to infer the physical state of Earth's upper mantle from seismic wave attenuation.
Timing of the Jiufotang Formation (Jehol Group) in Liaoning, northeastern China, and its implications
Authors: He H. Y. , Wang X. L. , Zhou Z. H. , Wang F. , Boven A. , Shi G. H. , Zhu R. X.
Published: June 2004

The timing of the Jiufotang Formation remains speculative despite recent progress in the study of the Jehol Biota. In this paper we contribute to this topic with 40Ar/39Ar dating on K‐feldspar (sanidine and orthoclase) from tuffs interbedded within the fossil‐bearing shales of the Jiufotang Formation, from the upper part of the Jehol Group in Chaoyang, Liaoning, northeastern China. 40Ar/39Ar step heating analyses of K‐feldspar and the SHRIMP U‐Pb zircon data indicate that tuffs at the Shangheshou section erupted at 120.3 ± 0.7 million years ago. This result confirms an Aptian age for the Jiufotang Formation that was mainly based on biostratigraphic evidence. It also places stringent controls on the age of the fossils from the formation, providing a minimum age (120 Ma) for the four‐winged dinosaur, Microraptor, and the seed‐eating bird, Jeholornis.
Implications of x‐ray emission from lightning
Authors: Dwyer J. R.
Published: June 2004

Dwyer et al. [2003, 2004a] recently reported measurements of bursts of x‐rays, with energies up to ∼250 keV, originating from dart leaders and possibly the return strokes of rocket‐triggered lightning. In this paper, these x‐ray observations are compared with the relativistic runaway electron avalanche (RREA) model. It is found that for dart leaders the standard RREA model is inconsistent with the observed spectrum and flux of the x‐ray emission. This result implies that the cold runaway electron model may be applicable to dart leaders. In this model, runaway electrons are directly accelerated out of the bulk electron population, produced by the ionization of the neutral atoms. However, this mechanism requires an electric field an order‐of‐magnitude larger than the conventional breakdown field. Because the flux of runaway electrons is very sensitive to the electric field strength, x‐ray observations of lightning provide a novel tool for determining the fields associated with the leaders.
Attenuation of 3,4‐methylenedioxymethamphetamine (MDMA, Ecstasy)‐induced rhabdomyolysis with α1‐ plus β3‐adrenoreceptor antagonists
Authors: Sprague Jon E , Brutcher Robert E , Mills Edward M , Caden David , Rusyniak Daniel E
Published: June 2004

Studies were designed to examine the effects of α1 (α1AR)‐ plus β3‐adrenoreceptor (β3AR) antagonists on 3,4‐methylenedioxymethamphetamine (MDMA, Ecstasy)‐induced hyperthermia and measures of rhabdomyolysis (creatine kinase (CK)) and renal function (blood urea nitrogen (BUN) and serum creatinine (sCr)) in male Sprague–Dawley rats.

MDMA (40 mg kg−1, s.c.) induced a rapid and robust increase in rectal temperature, which was significantly attenuated by pretreatment with the α1AR antagonist prazosin (100 μg kg−1, i.p.) plus the β3AR antagonist SR59230A (5 mg kg−1, i.p.).

CK levels significantly increased (peaking at 4 h) after MDMA treatment and were blocked by the combination of prazosin plus SR59230A.

At 4 h after MDMA treatment, BUN and sCr levels were also significantly increased and could be prevented by this combination of α1AR‐ plus β3AR‐antagonists.

The results from this study suggest that α1AR and β3AR play a critical role in the etiology of MDMA‐mediated hyperthermia and subsequent rhabdomyolysis.

British Journal of Pharmacology (2004) 142, 667–670. doi:10.1038/sj.bjp.0705823
Presynaptic cannabinoid CB1 receptors are involved in the inhibition of the neurogenic vasopressor response during septic shock in pithed rats
Authors: Godlewski Grzegorz , Malinowska Barbara , Schlicker Eberhard
Published: June 2004

Our study was undertaken to investigate whether bacterial endotoxin/lipopolysaccharide (LPS) affects the neurogenic vasopressor response in rats in vivo by presynaptic mechanisms and, if so, to characterize the type of presynaptic receptor(s) operating in the initial phase of septic shock.

In pithed and vagotomized rats treated with pancuronium, electrical stimulation (ES) (1 Hz, 1 ms, 50 V for 10 s) of the preganglionic sympathetic nerve fibers or intravenous bolus injection of noradrenaline (NA) (1–3 nmol kg−1) increased the diastolic blood pressure (DBP) by about 30 mmHg. Administration of LPS (0.4 and 4 mg kg−1) under continuous infusion of vasopressin inhibited the neurogenic vasopressor response by 25 and 50%, respectively. LPS did not affect the increase in DBP induced by exogenous NA.

The LPS‐induced inhibition of the neurogenic vasopressor response was counteracted by the cannabinoid CB1 receptor antagonist SR 141716A (0.1 μmol kg−1), but not by the CB2 receptor antagonist SR 144528 (3 μmol kg−1), the vanilloid VR1 receptor antagonist capsazepine (1 μmol kg−1) or the histamine H3 receptor antagonist clobenpropit (0.1 μmol kg−1). The four antagonists by themselves did not affect the increase in DBP induced by ES or by injection of NA in rats not exposed to LPS.

We conclude that in the initial phase of septic shock, the activation of presynaptic CB1 receptors by endogenously formed cannabinoids contributes to the inhibition of the neurogenic vasopressor response.

British Journal of Pharmacology (2004) 142, 701–708. doi:10.1038/sj.bjp.0705839
α1‐Adrenoceptor antagonist properties of CGP 12177A and other β‐adrenoceptor ligands: evidence against β3‐ or atypical β‐adrenoceptors in rat aorta
Authors: Brahmadevara Nataraj , Shaw Angus M , MacDonald Allan
Published: June 2004

The α1‐adrenoceptor antagonist properties of the β‐adrenoceptor nonconventional partial agonist, CGP 12177A, was investigated in functional assays in rat aorta and in radioligand binding assays in rat cerebral cortical membranes. In addition, binding affinities of other β‐adrenoceptor ligands were measured to investigate any correlation between α1‐adrenoceptor affinity and relaxant potency in phenylephrine‐constricted rings.

In functional studies, CGP 12177A produced parallel rightward shifts of the phenylephrine CRC with no reduction in the maximum responses. Schild regression analysis gave a straight line with a slope of 0.95 (95% CL: 0.87–1.04), suggesting reversible competitive antagonism, and gave a pKB value of 5.26. In contrast, CGP 12177A (300 μM) had no effect on contraction induced by the thromboxane‐mimetic, U46619.

In binding studies, CGP 12177A competed monophasically with [3H]prazosin binding (Hill slope, 0.95, 95% CL: 0.76–1.13), giving a pKi value of 5.48, in good agreement with the pKB from functional studies.

Competition experiments with various other β‐adrenoceptor ligands showed that they all displaced [3H]prazosin in a manner consistent with one‐site competition. pKi values were as follows: SR 59230A, 6.25; cyanopindolol, 6.33; bupranolol, 6.35; alprenolol, 5.90; propranolol, 5.80; BRL 37344, 5.50; ICI 118551, 5.55; CGP 20712A, 5.26. The pKi values correlated well with the pEC50 values for relaxation of phenylephrine‐constricted rat aorta obtained previously (r2=0.984, P<0.0001).

In conclusion, relaxant effects of CGP 12177A and other β‐adrenoceptor ligands in phenylephrine‐constricted rat aorta can be attributed to α1‐adrenoceptor blockade and are unrelated to effects at β3‐adrenoceptors or atypical β‐adrenoceptors.

British Journal of Pharmacology (2004) 142, 781–787. doi:10.1038/sj.bjp.0705840
Epidermal growth factor differentially augments Gi‐mediated stimulation of c‐Jun N‐terminal kinase activity
Authors: Chan Anthony S L , Wong Yung H
Published: June 2004

Signaling networks involving different receptor systems allow extracellular signals to be integrated and transformed into various biological activities. In this report, we studied the activity of the c‐Jun N‐terminal kinase (JNK) subgroup of mitogen‐activated protein kinases (MAPKs), in response to stimulation by G protein‐coupled receptors (GPCRs) and co‐activation with epithermal growth factor receptor (EGFR).

Stimulation of exogenous GPCRs in Cos‐7 cells induced JNK activation of different magnitudes depending on their G‐protein coupling specificities (Gq>Gi>Gs), and a moderate JNK activation was linked to stimulation of endogenous EGFR by EGF.

Co‐stimulation with GPCR agonists and EGF resulted in differential augmentation of JNK activities, with Gi‐coupled receptors associated with a synergistic JNK activation upon co‐stimulation with EGF, while Gq‐ and Gs‐coupled receptors were incapable of triggering this effect.

This Gi/EGF‐induced synergistic JNK activation was inhibited by pertussis toxin and AG1478, and may involve Src family tyrosine kinases, PI3 K, Ca2+/calmodulin and small GTPases as important intermediates, while Ca2+ mobilization was triggered by the stimulation of Gq‐coupled receptor or EGF treatment, but not by the Gi‐ or Gs‐coupled receptors.

Transient expression of Gβγ subunits with EGF treatment, or co‐activation of exogenous Gi‐coupled receptor with thapsigargin also resulted in a synergistic JNK activation. Activation of Gi‐coupled receptor accompanied with EGF treatment enhanced the expression level and activity of MAPK phosphatase type I, which occurred after the maximal synergistic JNK activation.

Our results support a mechanistic model where EGF signaling may differentially regulate the JNK activities triggered by GPCRs of different coupling specificities.

British Journal of Pharmacology (2004) 142, 635–646. doi:10.1038/sj.bjp.0705851
Differences in substrate and inhibitor sequence specificity of human, mouse and rat tissue kallikreins
Authors: FOGAÇA Sandro E. , MELO Robson L. , PIMENTA Daniel C. , HOSOI Kazuo , JULIANO Luiz , JULIANO Maria A.
Published: June 2004

The kininogenase activities of mouse (mK1), rat (rK1) and human (hK1) tissue kallikreins were assayed with the bradykinin-containing synthetic peptides Abz-MTEMARRPPGFSPFRSVTVQNH2 (where Abz stands for o-aminobenzoyl) and Abz-MTSVIRRPPGFSPFRAPRV-NH2, which correspond to fragments Met374-Gln393 and Met375-Val393 of mouse and rat LMWKs (low-molecular-mass kininogens) with the addition of Abz. Bradykinin was released from these peptides by the mK1- and rK1-mediated hydrolysis of Arg–Arg and Arg–Ser (or Arg–Ala) peptide bonds. However, owing to preferential hydrolysis of Phe–Arg compared with the Arg–Ala bond in the peptide derived from rat LMWK, hK1 released bradykinin only from the mouse LMWK fragment and preferentially released des-[Arg9]bradykinin from the rat LMWK fragment (Abz-MTSVIRRPPGFSPFRAPRV-NH2). The formation of these hydrolysis products was examined in more detail by determining the kinetic parameters for the hydrolysis of synthetic, internally quenched fluorescent peptides containing six N- or C-terminal amino acids of bradykinin added to the five downstream or upstream residues of mouse and rat kininogens respectively. One of these peptides, Abz-GFSPFRAPRVQ-EDDnp (where EDDnp stands for ethylenediamine 2,4-dinitrophenyl), was preferentially hydrolysed at the Phe–Arg bond, confirming the potential des-[Arg9]bradykinin-releasing activity of hK1 on rat kininogen. The proline residue that is two residues upstream of bradykinin in rat kininogen is, in part, responsible for this pattern of hydrolysis, since the peptide Abz-GFSPFRASRVQ-EDDnp was preferentially cleaved at the Arg–Ala bond by hK1. Since this peptidase accepts the arginine or phenylalanine residue at its S1 subsite, this preference seems to be determined by the prime site of the substrates. These findings also suggested that the effects observed in rats overexpressing hK1 should consider the activation of B1 receptors by des-[Arg9]bradykinin. For further comparison, two short internally quenched fluorescent peptides that bind to hK1 with affinity in the nM range and some inhibitors described previously for hK1 were also assayed with mK1 and rK1.
Ca2+ waves in keratinocytes are transmitted to sensory neurons: the involvement of extracellular ATP and P2Y2 receptor activation
Authors: KOIZUMI Schuichi , FUJISHITA Kayoko , INOUE Kaori , SHIGEMOTO-MOGAMI Yukari , TSUDA Makoto , INOUE Kazuhide
Published: June 2004

ATP acts as an intercellular messenger in a variety of cells. In the present study, we have characterized the propagation of Ca2+ waves mediated by extracellular ATP in cultured NHEKs (normal human epidermal keratinocytes) that were co-cultured with mouse DRG (dorsal root ganglion) neurons. Pharmacological characterization showed that NHEKs express functional metabotropic P2Y2 receptors. When a cell was gently stimulated with a glass pipette, an increase in [Ca2+]i (intracellular Ca2+ concentration) was observed, followed by the induction of propagating Ca2+ waves in neighbouring cells in an extracellular ATP-dependent manner. Using an ATP-imaging technique, the release and diffusion of ATP in NHEKs were confirmed. DRG neurons are known to terminate in the basal layer of keratinocytes. In a co-culture of NHEKs and DRG neurons, mechanical-stimulation-evoked Ca2+ waves in NHEKs caused an increase in [Ca2+]i in the adjacent DRG neurons, which was also dependent on extracellular ATP and the activation of P2Y2 receptors. Taken together, extracellular ATP is a dominant messenger that forms intercellular Ca2+ waves in NHEKs. In addition, Ca2+ waves in NHEKs could cause an increase in [Ca2+]i in DRG neurons, suggesting a dynamic cross-talk between skin and sensory neurons mediated by extracellular ATP.
p300 relieves p53-evoked transcriptional repression of hypoxia-inducible factor-1 (HIF-1)
Authors: SCHMID Tobias , ZHOU Jie , KÖHL Roman , BRÜNE Bernhard
Published: June 2004

HIF-1 (hypoxia-inducible factor-1), a heterodimeric transcription factor comprising HIF-1α and HIF-1β subunits, serves as a key regulator of metabolic adaptation to hypoxia. HIF-1 activity largely increases during hypoxia by attenuating pVHL (von Hippel–Lindau protein)-dependent ubiquitination and subsequent 26 S-proteasomal degradation of HIF-1α. Besides HIF-1, the transcription factor and tumour suppressor p53 accumulates and is activated under conditions of prolonged/severe hypoxia. Recently, the interaction between p53 and HIF-1α was reported to evoke HIF-1α degradation. Destruction of HIF-1α by p53 was corroborated in the present study by using pVHL-deficient RCC4 (renal carcinoma) cells, supporting the notion of a pVHL-independent degradation process. In addition, low p53 expression repressed HIF-1 transactivation without affecting HIF-1α protein amount. Establishing that p53-evoked inhibition of HIF-1 reporter activity was relieved upon co-transfection of p300 suggested competition between p53 and HIF-1 for limiting amounts of the shared co-activator p300. This assumption was confirmed by showing competitive binding of in vitro transcription/translation-generated p53 and HIF-1α to the CH1 domain of p300 in vitro. We conclude that low p53 expression attenuates HIF-1 transactivation by competing for p300, whereas high p53 expression destroys the HIF-1α protein and thereby eliminates HIF-1 reporter activity. Thus once p53 becomes activated under conditions of severe hypoxia/anoxia, it contributes to terminating HIF-1 responses.
Phosphorylation of clock protein PER1 regulates its circadian degradation in normal human fibroblasts
Authors: MIYAZAKI Koyomi , NAGASE Takahiro , MESAKI Miho , NARUKAWA Junko , OHARA Osamu , ISHIDA Norio
Published: June 2004

Recent advances suggest that the molecular components of the circadian clock generate a self-sustaining transcriptional–translational feedback loop with a period of approx. 24 h. The precise expression profiles of human clock genes and their products have not been elucidated. We cloned human clock genes, including per1, per2, per3, cry2 and clock, and evaluated their circadian mRNA expression profiles in WI-38 fibroblasts stimulated with serum. Transcripts of hPer1, hPer2, hPer3, hBMAL1 and hCry2 (where h is human) underwent circadian oscillation. Serum-stimulation also caused daily oscillations of hPER1 protein and the apparent molecular mass of hPER1 changed. Inhibitor studies indicated that the CKI (casein kinase I) family, including CKI∊ and CKIδ, phosphorylated hPER1 and increased the apparent molecular mass of hPER1. The inhibition of hPER1 phosphorylation by CKI-7 [N-(2-aminoethyl)-5-chloro-isoquinoline-8-sulphonamide], a CKI inhibitor, disturbed hPER1 degradation, delayed the nuclear entry of hPER1 and allowed it to persist for longer in the nucleus. Furthermore, proteasome inhibitors specifically blocked hPER1 degradation. However leptomycin B, an inhibitor of nuclear export, did not alter the degradation state of hPER1 protein. These findings indicate that circadian hPER1 degradation through a proteasomal pathway can be regulated through phosphorylation by CKI, but not by subcellular localization.
Isolation and characterization of lipid rafts with different properties from RBL-2H3 (rat basophilic leukaemia) cells
Authors: RADEVA Galina , SHAROM Frances J.
Published: June 2004

Lipid rafts are plasma-membrane microdomains that are enriched in certain lipids (sphingolipids, glycosphingolipids and cholesterol), as well as in lipid-modified proteins. Rafts appear to exist in the liquid-ordered phase, which contributes to their partitioning from the surrounding liquid-disordered glycerophospholipid environment. DRM (detergent-resistant membrane) fractions isolated from cells are believed to represent coalesced lipid rafts. We have employed extraction using two different non-ionic detergents, Brij-96 and Triton X-100, to isolate detergent-resistant lipid rafts from rat basophilic leukaemia cell line RBL-2H3, and compared their properties with each other and with plasma-membrane vesicles. DRM fractions were isolated as sealed unilamellar vesicles of similar size (135–170 nm diameter), using either sucrose-density-gradient sedimentation or gel-filtration chromatography. Lipid rafts isolated using Brij-96 and Triton X-100 differed in density, protein content and the distribution between high- and low-density fractions of the known raft constituents, Thy-1, and the non-receptor protein tyrosine kinases, Yes and Lyn. Lyn was found in the raft microdomains in predominantly phosphorylated form. The level of enrichment of the protein constituents of the isolated lipid rafts seemed to depend on the ratio of cell lipid/protein to detergent. As indicated by reactivity with anti-Thy-1 antibodies, lipid rafts prepared using Brij-96 appeared to consist of vesicles with primarily right-side-out orientation. Both Brij-96 and Triton X-100 appear to isolate detergent-insoluble raft microdomains from the rat basophilic leukaemia cell line RBL-2H3, but the observed differences suggest that either the detergents themselves play a role in determining the physicochemical characteristics of the resulting DRM fractions, or different subsets of rafts are isolated by the two detergents.
Thermodynamic characterization of monomeric and dimeric forms of CcdB (controller of cell division or death B protein)
Authors: BAJAJ Kanika , CHAKSHUSMATHI Ghadiyaram , BACHHAWAT-SIKDER Kiran , SUROLIA Avadhesha , VARADARAJAN Raghavan
Published: June 2004

The protein CcdB (controller of cell division or death B) is an F-plasmid-encoded toxin that acts as an inhibitor of Escherichia coli DNA gyrase. The stability and aggregation state of CcdB have been characterized as a function of pH and temperature. Size-exclusion chromatography revealed that the protein is a dimer at pH 7.0, but a monomer at pH 4.0. CD analysis and fluorescence spectroscopy showed that the monomer is well folded, and has similar tertiary structure to the dimer. Hence intersubunit interactions are not required for folding of individual subunits. The stability of both forms was characterized by isothermal denaturant unfolding and calorimetry. The free energies of unfolding were found to be 9.2 kcal·mol−1 (1 cal≈4.184 J) and 21 kcal·mol−1 at 298 K for the monomer and dimer respectively. The denaturant concentration at which one-half of the protein molecules are unfolded (Cm) of the dimer is dependent on protein concentration, whereas the Cm of the monomer is independent of protein concentration, as expected. Although thermal unfolding of the protein in aqueous solution is irreversible at neutral pH, it was found that thermal unfolding is reversible in the presence of GdmCl (guanidinium chloride). Differential scanning calorimetry in the presence of low concentrations of GdmCl in combination with isothermal denaturation melts as a function of temperature were used to derive the stability curve for the protein. The value of ΔCp (representing the change in excess heat capacity upon protein denaturation) is 2.8±0.2 kcal·mol−1·K−1 for unfolding of dimeric CcdB, and only has a weak dependence on denaturant concentration.
Stress- and mitogen-induced phosphorylation of the synapse-associated protein SAP90/PSD-95 by activation of SAPK3/p38gamma and ERK1/ERK2
Authors: SABIO Guadalupe , REUVER Suzana , FEIJOO Carmen , HASEGAWA Masato , THOMAS Gareth M. , CENTENO Francisco , KUHLENDAHL Sven , LEAL-ORTIZ Sergio , GOEDERT Michel , GARNER Craig , CUENDA Ana
Published: June 2004

SAPK3 (stress-activated protein kinase-3, also known as p38γ) is a member of the mitogen-activated protein kinase family; it phosphorylates substrates in response to cellular stress, and has been shown to bind through its C-terminal sequence to the PDZ domain of α1-syntrophin. In the present study, we show that SAP90 [(synapse-associated protein 90; also known as PSD-95 (postsynaptic density-95)] is a novel physiological substrate for both SAPK3/p38γ and the ERK (extracellular-signal-regulated protein kinase). SAPK3/p38γ binds preferentially to the third PDZ domain of SAP90 and phosphorylates residues Thr287 and Ser290in vitro, and Ser290 in cells in response to cellular stresses. Phosphorylation of SAP90 is dependent on the binding of SAPK3/p38γ to the PDZ domain of SAP90. It is not blocked by SB 203580, which inhibits SAPK2a/p38α and SAPK2b/p38β but not SAPK3/p38γ, or by the ERK pathway inhibitor PD 184352. However, phosphorylation is abolished when cells are treated with a cell-permeant Tat fusion peptide that disrupts the interaction of SAPK3/p38γ with SAP90. ERK2 also phosphorylates SAP90 at Thr287 and Ser290in vitro, but this does not require PDZ-dependent binding. SAP90 also becomes phosphorylated in response to mitogens, and this phosphorylation is prevented by pretreatment of the cells with PD 184352, but not with SB 203580. In neurons, SAP90 and SAPK3/p38γ co-localize and they are co-immunoprecipitated from brain synaptic junctional preparations. These results demonstrate that SAP90 is a novel binding partner for SAPK3/p38γ, a first physiological substrate described for SAPK3/p38γ and a novel substrate for ERK1/ERK2, and that phosphorylation of SAP90 may play a role in regulating protein–protein interactions at the synapse in response to adverse stress- or mitogenrelated stimuli.
Concentration-dependent effects of endogenous S-nitrosoglutathione on gene regulation by specificity proteins Sp3 and Sp1
Authors: ZAMAN Khalequz , PALMER Lisa A. , DOCTOR Allan , HUNT John F. , GASTON Benjamin
Published: June 2004

The activities of certain nuclear regulatory proteins are modified by high concentrations of S-nitrosothiols associated with nitrosative stress. In the present study, we have studied the effect of physiological (low µM) concentrations of the endogenous S-nitrosothiol, GSNO (S-nitrosoglutathione), on the activities of nuclear regulatory proteins Sp3 and Sp1 (specificity proteins 3 and 1). Low concentrations of GSNO increased Sp3 binding, as well as Sp3-dependent transcription of the cystic fibrosis transmembrane conductance regulatory gene, cftr. However, higher GSNO levels prevented Sp3 binding, augmented Sp1 binding and prevented both cftr transcription and CFTR (cystic fibrosis transmembrane conductance regulator) expression. We conclude that low concentrations of GSNO favour Sp3 binding to ‘housekeeping’ genes such as cftr, whereas nitrosative stress-associated GSNO concentrations shut off Sp3-dependent transcription, possibly to redirect cellular resources. Since low micromolar concentrations of GSNO also increase the maturation and activity of a clinically common CFTR mutant, whereas higher concentrations have the opposite effect, these observations may have implications for dosing of S-nitrosylating agents used in cystic fibrosis clinical trials.
Cysteine residues in the organic anion transporter mOAT1
Authors: TANAKA Kunihiko , ZHOU Fanfan , KUZE Kogo , YOU Guofeng
Published: June 2004

Mouse organic anion transporter 1 (mOAT1) belongs to a family of organic anion transporters, which play critical roles in the body disposition of clinically important drugs, including anti-HIV therapeutics, anti-tumour drugs, antibiotics, anti-hypertensives and anti-inflammatories. mOAT1-mediated transport of organic anion PAH (p-aminohippurate) in HeLa cells was inhibited by the cysteine-modifying reagent PCMBS (p-chloromercuribenzenesulphonate). Therefore the role of cysteine residues in the function of mOAT1 was examined by site-directed mutagenesis. All 13 cysteine residues in mOAT1 were replaced by alanine, singly or in combination. Single replacement of these residues had no significant effect on mOAT1-mediated PAH transport, indicating that no individual cysteine residue is necessary for function. Multiple replacements at a C-terminal region (C335/379/427/434A; Cys335/379/427/434→Ala) resulted in a substantial decrease in transport activity. A simultaneous replacement of all 13 cysteine residues (C-less) led to a complete loss of transport function. The decreased or lack of transport activity of the mutants C335/379/427/434A and C-less was due to the impaired trafficking of the mutant transporters to the cell surface. These results suggest that although cysteine residues are not required for function in mOAT1, their presence appears to be important for the targeting of the transporter to the plasma membrane. We also showed that, although all cysteine mutants of mOAT1 were sensitive to the inhibition by PCMBS, C49A was less sensitive than the wild-type mOAT1, suggesting that the modification of Cys49 may play a role in the inhibition of mOAT1 by PCMBS.
Ceramide metabolite, not intact ceramide molecule, may be responsible for cellular toxicity
Authors: TSERNG Kou-Yi , GRIFFIN Ronda L.
Published: June 2004

Ceramides, which are produced from the hydrolysis of sphingomyelin or synthesized from serine and palmitate in a de novo pathway, are regarded as important cellular signals for inducing apoptosis. However, controversy over this proposed role of ceramides exists. Using stable isotope labelling coupled with GC (gas chromatography)-MS and mass isotopomer distribution analysis, we have studied the metabolism of exogenous long-chain ceramides in HL60 cells. Our results do not support the concept of enhanced ceramide transport into cells induced by solvent mixtures of ethanol and hydrocarbons. In addition, cell toxicity does not correlate with the amount of intact ceramide in the cells. Our results are more consistent with a disturbance of sphingomyelin metabolism induced by the solvent mixture. The characteristics of this disturbed sphingolipid disposition are the inhibition of dihydroceramide desaturation and an enhanced degradation of sphingomyelin. As a consequence, dihydroceramides accumulate and the cellular sphingomyelin content decreases. Inhibition of these pathways is most likely to be induced by the increased production of novel ceramide metabolites instead of by intact ceramides. Octadecane-1,2-diol is identified as a possible mediator. Treatments that divert ceramide degradation to the novel pathway are potential strategies in cancer therapy for inducing cell toxicity.
Oligomerization of bovine ribonuclease A: structural and functional features of its multimers
Authors: LIBONATI Massimo , GOTTE Giovanni
Published: June 2004

Bovine pancreatic RNase A (ribonuclease A) aggregates to form various types of catalytically active oligomers during lyophilization from aqueous acetic acid solutions. Each oligomeric species is present in at least two conformational isomers. The structures of two dimers and one of the two trimers have been solved, while plausible models have been proposed for the structures of a second trimer and two tetrameric conformers. In this review, these structures, as well as the general conditions for RNase A oligomerization, based on the well known 3D (three-dimensional) domain-swapping mechanism, are described and discussed. Attention is also focused on some functional properties of the RNase A oligomers. Their enzymic activities, particularly their ability to degrade double-stranded RNAs and polyadenylate, are summarized and discussed. The same is true for the remarkable antitumour activity of the oligomers, displayed in vitro and in vivo, in contrast with monomeric RNase A, which lacks these activities. The RNase A multimers also show an aspermatogenic action, but lack any detectable embryotoxicity. The fact that both activity against double-stranded RNA and the antitumour action increase with the size of the oligomer suggests that these activities may share a common structural requirement, such as a high number or density of positive charges present on the RNase A oligomers.
Cloning, tissue distribution, pharmacology and three-dimensional modelling of melanocortin receptors 4 and 5 in rainbow trout suggest close evolutionary relationship of these subtypes
Authors: HAITINA Tatjana , KLOVINS Janis , ANDERSSON Jan , FREDRIKSSON Robert , LAGERSTRÖM Malin C. , LARHAMMAR Dan , LARSON Earl T. , SCHIÖTH Helgi B.
Published: June 2004

The rainbow trout (Oncorhynchus mykiss) is one of the most widely used fish species in aquaculture and physiological research. In the present paper, we report the first cloning, 3D (three-dimensional) modelling, pharmacological characterization and tissue distribution of two melanocortin (MC) receptors in rainbow trout. Phylogenetic analysis indicates that these receptors are orthologues of the human MC4 and MC5 receptors. We created 3D molecular models of these rainbow trout receptors and their human counterparts. These models suggest greater divergence between the two human receptors than between their rainbow trout counterparts. The pharmacological analyses demonstrated that ACTH (adrenocorticotropic hormone) had surprisingly high affinity for the rainbow trout MC4 and MC5 receptors, whereas α-, β- and γ-MSH (melanocyte-stimulating hormone) had lower affinity. In second-messenger studies, the cyclic MSH analogues MTII and SHU9119 acted as potent agonist and antagonist respectively at the rainbow trout MC4 receptor, indicating that these ligands are suitable for physiological studies in rainbow trout. Interestingly, we found that the rainbow trout MC4 receptor has a natural high-affinity binding site for zinc ions (0.5 µM) indicating that zinc may play an evolutionary conserved role at this receptor. Reverse transcription PCR indicates that the rainbow trout receptors are expressed both in peripheral tissues and in the central nervous system, including the telencephalon, optic tectum and hypothalamus. Overall, this analysis indicates that the rainbow trout MC4 and MC5 receptors have more in common than their mammalian counterparts, which may suggest that these two receptors have a closer evolutionary relationship than the other MC receptor subtypes.
Acyl-CoA-binding protein, Acb1p, is required for normal vacuole function and ceramide synthesis in Saccharomyces cerevisiae
Authors: FÆRGEMAN Nils J. , FEDDERSEN Søren , CHRISTIANSEN Janne K. , LARSEN Morten K. , SCHNEITER Roger , UNGERMANN Christian , MUTENDA Kudzai , ROEPSTORFF Peter , KNUDSEN Jens
Published: June 2004

In the present study, we show that depletion of acyl-CoA-binding protein, Acb1p, in yeast affects ceramide levels, protein trafficking, vacuole fusion and structure. Vacuoles in Acb1p-depleted cells are multi-lobed, contain significantly less of the SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) Nyv1p, Vam3p and Vti1p, and are unable to fuse in vitro. Mass spectrometric analysis revealed a dramatic reduction in the content of ceramides in whole-cell lipids and in vacuoles isolated from Acb1p-depleted cells. Maturation of yeast aminopeptidase I and carboxypeptidase Y is slightly delayed in Acb1p-depleted cells, whereas the maturation of alkaline phosphatase and Gas1p is unaffected. The fact that Gas1p maturation is unaffected by Acb1p depletion, despite the lowered ceramide content in these cells, indicates that ceramide synthesis in yeast could be compartmentalized. We suggest that the reduced ceramide synthesis in Acb1p-depleted cells leads to severely altered vacuole morphology, perturbed vacuole assembly and strong inhibition of homotypic vacuole fusion.
Secondary-structure characterization by far-UV CD of highly purified uncoupling protein 1 expressed in yeast
Authors: DOUETTE Pierre , NAVET Rachel , BOUILLENNE Fabrice , BRANS Alain , SLUSE-GOFFART Claudine , MATAGNE André , SLUSE Francis E.
Published: June 2004

The rat UCP1 (uncoupling protein 1) is a mitochondrial inner-membrane carrier involved in energy dissipation and heat production. We expressed UCP1 carrying a His6 epitope at its C-terminus in Saccharomyces cerevisiae mitochondria. The recombinant-tagged UCP1 was purified by immobilized metal-ion affinity chromatography to homogeneity (>95%). This made it suitable for subsequent biophysical characterization. Fluorescence resonance energy transfer experiments showed that n-dodecyl-β-d-maltoside-solubilized UCP1–His6 retained its PN (purine nucleotide)-binding capacity. The far-UV CD spectrum of the functional protein clearly indicated the predominance of α-helices in the UCP1 secondary structure. The UCP1 secondary structure exhibited an α-helical degree of approx. 68%, which is at least 25% higher than the previously reported estimations based on computational predictions. Moreover, the helical content remained unchanged in free and PN-loaded UCP1. A homology model of the first repeat of UCP1, built on the basis of X-ray-solved close parent, the ADP/ATP carrier, strengthened the CD experimental results. Our experimental and computational results indicate that (i) α-helices are the major component of UCP1 secondary structure; (ii) PN-binding mechanism does not involve significant secondary-structure rearrangement; and (iii) UCP1 shares similar secondary-structure characteristics with the ADP/ATP carrier, at least for the first repeat.
Expression and characterization of recombinant fungal acetyl-CoA carboxylase and isolation of a soraphen-binding domain
Authors: WEATHERLY Stephanie C. , VOLRATH Sandra L. , ELICH Tedd D.
Published: June 2004

Acetyl-CoA carboxylase (ACC) catalyses the first step in fatty-acid biosynthesis. Owing to its role in primary metabolism, ACC has been exploited as a commercial herbicide target and identified as a chemically validated fungicide target. In animals, ACC is also a key regulator of fat metabolism. This function has made ACC a prime target for the development of anti-obesity and anti-Type II diabetes therapeutics. Despite its economic importance, there is a lack of published information on recombinant expression of ACC. We report here the expression of enzymically active fungal (Ustilago maydis) ACC in Escherichia coli. The recombinant enzyme exhibited Km values of 0.14±0.013 mM and 0.19±0.041 mM for acetyl-CoA and ATP respectively, which are comparable with those reported for the endogenous enzyme. The polyketide natural product soraphen is a potent inhibitor of the BC (biotin carboxylase) domain of endogenous fungal ACC. Similarly, recombinant ACC activity was inhibited by soraphen with a Ki of 2.1±0.9 nM. A truncated BC domain that included amino acids 2–560 of the full-length protein was also expressed in E. coli. The isolated BC domain was expressed to higher levels, and was more stable than full-length ACC. Although incapable of enzymic turnover, the BC domain exhibited high-affinity soraphen binding (Kd 1.1±0.3 nM), demonstrating a native conformation. Additional BC domains from the phytopathogenic fungi Magnaporthe grisea and Phytophthora infestans were also cloned and expressed, and were shown to exhibit high-affinity soraphen binding. Together, these reagents will be useful for structural studies and assay development.
Novel activating and inactivating mutations in the integrin beta1 subunit A domain
Authors: BARTON Stephanie J. , TRAVIS Mark A. , ASKARI Janet A. , BUCKLEY Patrick A. , CRAIG Susan E. , HUMPHRIES Martin J. , MOULD A. Paul
Published: June 2004

The ligand-binding activity of integrins is regulated by shape changes that convert these receptors from a resting (or inactive) state to an active state. However, the precise conformational changes that take place in head region of integrins (the site of ligand binding) during activation are not well understood. The portion of the integrin β subunit involved in ligand recognition contains a von Willebrand factor type A domain, which comprises a central β-sheet surrounded by seven α helices (α1–α7). Using site-directed mutagenesis, we show here that point mutation of hydrophobic residues in the α1 and α7 helices (which would be predicted to increase the mobility of these helices) markedly increases the ligand-binding activity of both integrins α5β1 and α4β1. In contrast, mutation of a hydrophilic residue near the base of the α1 helix decreases activity and also suppresses exposure of activation epitopes on the underlying hybrid domain. Our results provide new evidence that shifts of the α1 and α7 helices are involved in activation of the A domain. Although these changes are grossly similar to those defined in the A domains found in some integrin α subunits, movement of the α1 helix appears to play a more prominent role in βA domain activation.
The shed ectodomain of type XIII collagen affects cell behaviour in a matrix-dependent manner
Authors: VÄISÄNEN Marja-Riitta , VÄISÄNEN Timo , PIHLAJANIEMI Taina
Published: June 2004

Transmembrane type XIII collagen resides in adhesive structures of cells and tissues, and has therefore been implicated in cell adhesion and in adhesion-dependent cell functions. This collagen also exists as a soluble protein in the pericellular matrix, as the ectodomain is released from the plasma membrane by proteolytic cleavage. Analysis with various protease inhibitors led to confirmation of the furin family of proprotein convertases as the protease group responsible for the shedding of the ectodomain, cleaving at a site conforming to the consensus sequence for the proprotein convertases at the stem of the ectodomain. Both the trans-Golgi network and the plasma membrane were used as cleavage locations. Mammalian cells employed various intracellular mechanisms to modulate shedding of the ectodomain, all resulting in a similar cleavage event. Cell detachment from the underlying substratum was also found to augment the excision. The released ectodomain rendered the pericellular surroundings less supportive of cell adhesion, migration and proliferation, as seen specifically on a vitronectin substratum. Type XIII collagen ectodomain shedding thus resulted in the formation of a soluble, biologically active molecule, which eventually modulated cell behaviour in a reciprocal and substratum-specific manner. The dual existence of membrane-bound and soluble variants widens our biological understanding of type XIII collagen.
Increased levels of insulin and insulin-like growth factor-1 hybrid receptors and decreased glycosylation of the insulin receptor alpha- and beta-subunits in scrapie-infected neuroblastoma N2a cells
Authors: NIELSEN Daniel , GYLLBERG Hanna , ÖSTLUND Pernilla , BERGMAN Tomas , BEDECS Katarina
Published: June 2004

We have previously shown that ScN2a cells (scrapie-infected neuroblastoma N2a cells) express 2-fold- and 4-fold-increased levels of IR (insulin receptor) and IGF-1R (insulin-like growth factor-1 receptor) respectively. In addition, the IR α- and β-subunits are aberrantly processed, with apparent molecular masses of 128 and 85 kDa respectively, as compared with 136 and 95 kDa in uninfected N2a cells. Despite the 2-fold increase in IR protein, the number of 125I-insulin-binding sites was slightly decreased in ScN2a cells [Östlund, Lindegren, Pettersson and Bedecs (2001) Brain Res. 97, 161–170]. In order to determine the cellular localization of IR in ScN2a cells, surface biotinylation was performed, showing a correct IR trafficking and localization to the cell surface. The present study shows for the first time that neuroblastoma N2a cells express significant levels of IR–IGF-1R hybrid receptors, and in ScN2a cells the number of hybrid receptors was 2-fold higher than that found in N2a cells, potentially explaining the apparent loss of insulin-binding sites due to a lower affinity for insulin compared with the homotypic IR. Furthermore, the decreased molecular mass of IR subunits in ScN2a cells is not caused by altered phosphorylation or proteolytic processing, but rather by altered glycosylation. Enzymic deglycosylation of immunoprecipitated IR from N2a and ScN2a cells with endoglycosidase H, peptide N-glycosidase F and neuraminidase all resulted in subunits with increased electrophoretic mobility; however, the 8–10 kDa shift remained. Combined enzymic or chemical deglycosylation using anhydrous trifluoromethane sulphonic acid treatment ultimately showed that the IR α- and β-subunits from ScN2a cells are aberrantly glycosylated. The increased formation of IR–IGF-1R hybrids in ScN2a cells may be part of a neuroprotective response to prion infection. The degree and functional significance of aberrantly glycosylated proteins in ScN2a cells remain to be determined.
Dual targeting of yeast catalase A to peroxisomes and mitochondria
Authors: PETROVA Ventsislava Y. , DRESCHER Diane , KUJUMDZIEVA Anna V. , SCHMITT Manfred J.
Published: June 2004

Yeast catalase A (Cta1p) contains two peroxisomal targeting signals (SSNSKF) localized at its C-terminus and within the N-terminal third of the protein, which both can target foreign proteins to peroxisomes. In the present study we demonstrated that Cta1p can also enter mitochondria, although the enzyme lacks a classical mitochondrial import sequence. Cta1p co-targeting was studied in a catalase A null mutant after growth on different carbon sources, and expression of a Cta1p–GFP (green fluorescent protein)-fusion protein or a Cta1p derivative containing either a c-Myc epitope (Cta1pmyc) or a SKF-extended tag (Cta1pmyc-SKF). Peroxisomal and mitochondrial co-import of catalase A were tested qualitatively by fluorescence microscopy and functional complementation of a Δcta1 null mutation, and quantitatively by subcellular fractionation followed by Western blot analysis and enzyme activity assays. Efficient Cta1p import into peroxisomes was observed when cells were cultivated under peroxisome-inducing conditions (i.e. growth on oleate), whereas significant co-import of Cta1p–GFP into mitochondria occurred when cells were grown under respiratory conditions that favour oxygen stress and ROS (reactive oxygen species) accumulation within this organelle. In particular, when cells were grown on the non-fermentable carbon source raffinose, respiration is maximally enhanced, and catalase A was efficiently targeted to the mitochondrial matrix where it presumably functions as scavenger of H2O2 and mitochondrial-derived ROS.
Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts
Authors: HUTTER Eveline , RENNER Kathrin , PFISTER Gerald , STÖCKL Petra , JANSEN-DÜRR Pidder , GNAIGER Erich
Published: June 2004

Limitation of lifespan in replicative senescence is related to oxidative stress, which is probably both the cause and consequence of impaired mitochondrial respiratory function. The respiration of senescent human diploid fibroblasts was analysed by highresolution respirometry. To rule out cell-cycle effects, proliferating and growth-arrested young fibroblasts were used as controls. Uncoupled respiration, as normalized to citrate synthase activity, remained unchanged, reflecting a constant capacity of the respiratory chain. Oligomycin-inhibited respiration, however, was significantly increased in mitochondria of senescent cells, indicating a lower coupling of electron transport with phosphorylation. In contrast, growth-arrested young fibroblasts exhibited a higher coupling state compared with proliferating controls. In intact cells, partial uncoupling may lead to either decreased oxidative ATP production or a compensatory increase in routine respiration. To distinguish between these alternatives, we subtracted oligomycin-inhibited respiration from routine respiration, which allowed us to determine the part of respiratory activity coupled with ATP production. Despite substantial differences in the respiratory control ratio, ranging from 4 to 11 in the different experimental groups, a fixed proportion of respiratory capacity was maintained for coupled oxidative phosphorylation in all the experimental groups. This finding indicates that the senescent cells fully compensate for increased proton leakage by enhanced electron-transport activity in the routine state. These results provide a new insight into age-associated defects in mitochondrial function and compensatory mechanisms in intact cells.
RNA interference-mediated silencing of synaptotagmin IX, but not synaptotagmin I, inhibits dense-core vesicle exocytosis in PC12 cells
Authors: FUKUDA Mitsunori
Published: June 2004

Although PC12 cells express three synaptotagmin isoforms (Syts I, IV and IX), all of which have been proposed to regulate dense-core vesicle exocytosis, it remains unknown which of the Sytisoforms acts as the major Ca2+ sensor for dense-core vesicle exocytosis. In the present study, it has been shown by immunoaffinity purification and immunocytochemistry that Syts I and IX, but not Syt IV, are present on the same secretory vesicles in PC12 cells. Silencing of Syt IX with specific small interfering RNA significantly reduced high KCl-dependent neuropeptide Y secretion from PC12 cells, whereas silencing of Syt I with specific small interfering RNA had no significant effect. The results indicate that Syts I and IX are not functionally equivalent and that Syt IX, and not Syt I, is indispensable for the regulation of Ca2+-dependent dense-core vesicle exocytosis in PC12 cells.