bluuebunny/crossref_metadata_2025 huggingface dataset:
Linkages between turbidites in the southern Okinawa Trough and submarine earthquakes
Authors: Huh Chih‐An , Su Chih‐Chieh , Liang Wen‐Tzong , Ling Chiao‐Yuen
Published: June 2004

Turbidite layers in surficial (<0.4 m) sediments of the southern Okinawa Trough and its vicinity were dated by 210Pb and further constrained by 137Cs and inter‐site correlation of downcore profiles of particle size and porosity. Here we show striking temporal and spatial correspondence of such episodic deposits to large (ML > 6.8) submarine earthquakes recorded in the region since the 20th century. The repeating pattern of turbidite layers reported here on decadal to centennial time scales suggests what may be the long‐term rhythm of seismic activities at this tectonically active plate boundary.
The Eddy Experiment: Accurate GNSS‐R ocean altimetry from low altitude aircraft
Authors: Ruffini G. , Soulat F. , Caparrini M. , Germain O. , Martín‐Neira M.
Published: June 2004

During the Eddy Experiment, two synchronous GPS receivers were flown at 1 km altitude to collect L1 signals and their reflections from the sea surface for assessment of altimetric precision and accuracy. Wind speed (U10) was around 10 m/s, and SWH up to 2 m. A geophysical parametric waveform model was used for retracking and estimation of the lapse between the direct and reflected signals with a 1‐second precision of 3 m. The lapse was used to estimate the SSH along the track using a differential model. The RMS error of the 20 km averaged GNSS‐R absolute altimetric solution with respect to Jason‐1 SSH and a GPS buoy measurement was of 10 cm, with a 2 cm mean difference. Multipath and retracking parameter sensitivity due to the low altitude are suspected to have degraded accuracy. This result provides an important milestone on the road to a GNSS‐R mesoscale altimetry space mission.
Exchange between Long Island Sound and adjacent waters
Authors: Gay Peter S. , O'Donnell James , Edwards Christopher A.
Published: June 2004

We exploit archived salinity data, averaged by month for the years 1995–2001, to evaluate exchange of salt between Long Island Sound and shelf waters, and treat Long Island Sound as a control volume with eastern and western boundaries at The Race and the East River, respectively. The use of inverse methods to solve a system of equations expressing conservation of salt and water provides estimates of model parameters together with uncertainty estimates and measures of model performance. We explore the use of a single exchange coefficient acting on an axial salinity gradient to represent exchange at The Race and find that it is necessary to allow the coefficient to depend weakly on variability of freshwater discharge, R, into the sound, i.e., = ()0.2(1,600 ± 200) m2/s. Using established estimates of the mean circulation at the western boundary of the sound, we determine a dispersion coefficient in the Upper East River of 300 ± 200 m2/s. Explicitly modeling the advective exchange flow (gravitational circulation) at The Race using a coefficient that varies as the one‐third power of the normalized discharge indicates that gravitational circulation accounts for less than 10% of the up‐estuary salt flux in eastern Long Island Sound.
Parameter and model sensitivities for colloid‐facilitated radionuclide transport on the field scale
Authors: Cvetkovic V. , Painter S. , Turner D. , Pickett D. , Bertetti P.
Published: June 2004

We investigate the potential effects of inorganic colloids on radionuclide transport in groundwater using generic sensitivity studies and an example based on the alluvial aquifer near Yucca Mountain, Nevada. Our emphasis is on kinetically controlled sorption of radionuclides on mobile and immobile colloids. Three kinetic sorption models are considered for the sensitivity analysis: bilinear, Langmuir, and linear. Plutonium is assumed to be injected into the Yucca Mountain alluvial aquifer at a constant rate and follows a random stream tube to a monitoring boundary. The linear sorption model provides a reasonable upper bound on colloid‐facilitated plutonium transport for the site‐specific conditions. In the absence of colloid filtration and retardation, colloids enhance the plutonium discharge by a large factor over the situation without colloids. Exchange of plutonium between solution and reversibly attached colloids makes colloid retardation relatively ineffective at reducing colloid‐facilitated transport except when the retardation factor is large. Irreversible removal of colloids (filtration) is more effective than retardation at reducing colloid‐facilitated transport. For fixed filtration rate the degree of attenuation depends sensitively and nonmonotonically on the rate of plutonium desorption from colloids. These results emphasize the need for accurate measurements of rates of desorption from colloids as well as in situ studies of filtration of naturally occurring colloids.
Auranofin induces apoptosis and when combined with retinoic acid enhances differentiation of acute promyelocytic leukaemia cells in vitro
Authors: Kim I S , Jin J Y , Lee I H , Park S J
Published: June 2004

Acute promyelocytic leukaemia (APL) is characterized by a block in differentiation at the promyelocyte stage. Here, we describe the effects of auranofin (AF), a coordinated gold compound, on apoptosis and differentiation of APL cells.

Nucleosomal DNA fragmentation assay and Hoechst 33342 staining indicated that AF induced apoptosis in APL‐derived NB4 cells at low concentrations (0.5–1.0 μM). The AF‐induced apoptosis involved caspase‐3 activation and specific cleavage of poly‐ADP‐ribose polymerase.

The AF‐treated NB4 cells also produced reactive oxygen species (ROS) and cotreatment with N‐acetyl‐L‐cysteine protected the NB4 cells from AF‐induced apoptosis.

Expression of the CD11b cell surface marker and C/EBPɛ was increased when the cells were treated for 4 days with 0.3 μM AF and a physiological concentration of all‐trans retinoic acid (ATRA, 5 nM). Treatment with AF in combination with ATRA markedly increased the number of cells with differentiated features, such as lobed or multiple nuclei and numerous granules and vacuoles. At these low concentrations, neither AF nor ATRA alone induced significant cell differentiation.

These findings suggest not only that AF induces caspase‐3‐dependent apoptosis via a mechanism involving ROS, but also that the combined treatment with AF and ATRA induces differentiation of NB4 cells. Our results demonstrate a novel characteristic of AF from which an effective drug treatment of APL might be developed.

British Journal of Pharmacology (2004) 142, 749–755. doi:10.1038/sj.bjp.0705708
Alpha‐1D adrenoceptors are involved in reserpine‐induced supersensitivity of rat tail artery
Authors: Taki Naoyuki , Tanaka Takashi , Zhang Li , Suzuki Fumiko , Israilova Malika , Taniguchi Takanobu , Hiraizumi‐Hiraoka Yasuko , Shinozuka Kazumasa , Kunitomo Masaru , Muramatsu Ikunobu
Published: June 2004

We examined reserpine‐induced chemical denervation supersensitivity with special reference to alpha‐1 adrenoceptor (AR) subtypes.

Chronic treatment with reserpine for 2 weeks depleted noradrenaline in the tail artery and spleen of rats. Noradrenaline in the thoracic aorta was negligible before and after reserpine treatment.

The treatment with reserpine produced supersensitivity in the contractile responses of the rat tail artery to phenylephrine, 5‐HT and KCl, resulting in leftward shift of concentration–response curves (11.6‐, 2.5‐ and 1.1‐fold at EC50 value, respectively). These results suggest a predominant sensitization of the alpha‐1 AR‐mediated response by reserpine treatment.

BMY 7378 at a concentration (30 nM) specific for blocking the alpha‐1D AR subtype, but not KMD‐3213 at a concentration (10 nM) selective for blocking the alpha‐1A AR subtype, inhibited the supersensitivity of the phenylephrine‐induced response in the reserpine‐treated artery. On the other hand, the response to phenylephrine in reserpine‐untreated artery was selectively inhibited by the same concentration of KMD‐3213, but not by BMY 7378. Prazosin, a subtype‐nonselective antagonist, blocked the responses to phenylephrine with the same potency, regardless of reserpine treatment.

In the thoracic aorta and spleen, no supersensitivity was produced in the responses to phenylephrine by reserpine treatment.

In a tissue segment‐binding study using [3H]‐prazosin, the total density and affinity of alpha‐1 ARs in the rat tail artery were not changed by treatment with reserpine. However, alpha‐1D AR with high affinity for BMY 7378 was significantly detected in reserpine‐treated tail artery, in contrast to untreated artery. Decreases in alpha‐1A AR with high affinity for KMD‐3213 and alpha‐1B AR with low affinities for KMD‐3213 and BMY 7378 were also estimated in reserpine‐treated tail artery.

Alpha‐1D AR mRNA in rat tail artery increased to three‐folds by reserpine treatment, whereas the levels of alpha‐1A and 1B mRNAs were not significantly changed.

The present results suggest that chronic treatment with reserpine affects the expression of alpha‐1 AR subtypes of rat tail artery and that the induction of alpha‐1D ARs with high affinity for catecholamines is in part associated with reserpine‐induced supersensitivity.

British Journal of Pharmacology (2004) 142, 647–656. doi:10.1038/sj.bjp.0705817
Comparative anti‐inflammatory activities of antagonists to C3a and C5a receptors in a rat model of intestinal ischaemia/reperfusion injury
Authors: Proctor Lavinia M , Arumugam Thiruma V , Shiels Ian , Reid Robert C , Fairlie David P , Taylor Stephen M
Published: June 2004

Complement activation is implicated in the pathogenesis of intestinal ischaemia–reperfusion injury (I/R), although the relative importance of individual complement components is unclear. A C3a receptor antagonist N(2)‐[(2,2‐diphenylethoxy)acetyl]‐L‐arginine (C3aRA) has been compared with a C5a receptor antagonist (C5aRA), AcF‐[OPdChaWR], in a rat model of intestinal I/R.

C3aRA (IC50=0.15 μM) and C5aRA (IC50=0.32 μM) bound selectively to human polymorphonuclear leukocyte (PMN) C3a and C5a receptors, respectively. Effects on circulating neutrophils and blood pressure in the rat were also assessed.

Anaesthetised rats, subjected to intestinal ischaemia (30 min) and reperfusion (120 min), were administered intravenously with either (A) the C3aRA (0.1–1.0 mg kg−1); the C5aRA (1.0 mg kg−1); the C3aRA+C5aRA (each 1.0 mg kg−1); or vehicle, 45 min prior, or (B) the C3aRA (1.0 mg kg−1) or vehicle, 120 min prior to reperfusion.

The C3aRA and C5aRA, administered 45 min prior to reperfusion, displayed similar efficacies at ameliorating several disease markers (increased oedema, elevated ALT levels and mucosal damage) of rat intestinal I/R. The combination drug treatment did not result in greater injury reduction than either antagonist alone. However, doses of the C3aRA (0.01–10 mg kg−1) caused transient neutropaenia, and the highest dose (10 mg kg−1) also caused a rapid and transient hypertension.

The C3aRA (1.0 mg kg−1), delivered 120 min prior to reperfusion to remove the global effect of C3aRA‐induced neutrophil sequestration, did not attenuate the markers of intestinal I/R, despite persistent C3aR antagonism at this time.

C3aR antagonism does not appear to be responsible for the anti‐inflammatory actions of this C3aRA in intestinal I/R in the rat. Instead, C3aRA‐mediated global neutrophil tissue sequestration during ischaemia and early reperfusion may account for the protective effects observed.

British Journal of Pharmacology (2004) 142, 756–764. doi:10.1038/sj.bjp.0705819
Mechanisms involved in carbachol‐induced Ca2+ sensitization of contractile elements in rat proximal and distal colon
Authors: Takeuchi Tadayoshi , Kushida Masahiko , Hirayama Nobue , Kitayama Muneyoshi , Fujita Akikazu , Hata Fumiaki
Published: June 2004

Mechanisms involved in Ca2+ sensitization of contractile elements induced by the activation of muscarinic receptors in membrane‐permeabilized preparations of the rat proximal and distal colon were studied.

In α‐toxin‐permeabilized preparations from the rat proximal and distal colon, Ca2+ induced a rapid phasic and subsequent tonic component. After Ca2+‐induced contraction reached a plateau, guanosine 5′‐triphosphate (GTP) and carbachol (CCh) in the presence of GTP further contracted preparations of both the proximal and distal colon (Ca2+ sensitization).

Y‐27632, a rho‐kinase inhibitor, inhibited GTP plus CCh‐induced Ca2+ sensitization more significantly in the proximal colon than in the distal colon. Y‐27632 at 10 μM had no effect on Ca2+‐induced contraction or slightly inhibited phorbol‐12,13‐dibutyrate‐induced Ca2+ sensitization in either proximal or distal colon. Chelerythrine, a protein kinase C inhibitor, inhibited GTP plus CCh‐induced Ca2+ sensitization in the distal colon, but not in the proximal colon. The component of Ca2+ sensitization that persisted after the chelerythrine treatment was completely inhibited by Y‐27632.

In β‐escin‐permeabilized preparations of the proximal colon, C3 exoenzyme completely inhibited GTP plus CCh‐induced Ca2+ sensitization, but PKC(19–31) did not. In the distal colon, C3 exoenzyme abolished GTP‐induced Ca2+ sensitization. It inhibited CCh‐induced sensitization by 50 % and the remaining component was inhibited by PKC(19–31).

These results suggest that both protein kinase C and rho pathways in parallel mediate the Ca2+ sensitization coupled to activation of muscarinic receptors in the rat distal colon, whereas the rho pathway alone mediates this action in the proximal colon.

British Journal of Pharmacology (2004) 142, 657–666. doi:10.1038/sj.bjp.0705820
Acute and late effects on induction of allodynia by acromelic acid, a mushroom poison related structurally to kainic acid
Authors: Minami Toshiaki , Matsumura Shinji , Nishizawa Mikio , Sasaguri Yasuyuki , Hamanaka Nobuyuki , Ito Seiji
Published: June 2004

Ingestion of a poisonous mushroom Clitocybe acromelalga is known to cause severe tactile pain (allodynia) in the extremities for a month and acromelic acid (ACRO), a kainate analogue isolated from the mushroom, produces selective damage of interneurons of the rat lower spinal cord when injected either systemically or intrathecally. Since ACRO has two isomers, ACRO‐A and ACRO‐B, here we examined their acute and late effects on induction of allodynia.

Intrathecal administration of ACRO‐A and ACRO‐B provoked marked allodynia by the first stimulus 5 min after injection, which lasted over the 50‐min experimental period. Dose‐dependency of the acute effect of ACRO‐A on induction of allodynia showed a bell‐shaped pattern from 50 ag kg−1 to 0.5 pg kg−1 and the maximum effect was observed at 50 fg kg−1. On the other hand, ACRO‐B induced allodynia in a dose‐dependent manner from 50 pg kg−1 to 50 ng kg−1.

N‐methyl‐D‐aspartate (NMDA) receptor antagonists and Joro spider toxin, a Ca2+‐permeable AMPA receptor antagonist, inhibited the allodynia induced by ACRO‐A, but not by ACRO‐B. However, other AMPA/kainate antagonists did not affect the allodynia induced by ACRO.

Whereas no neuronal damage was observed in the spinal cord in ACRO‐A‐treated mice, induction of allodynia by ACRO‐A (50 fg kg−1) and ACRO‐B (50 ng kg−1) was selectively lost 1 week after i.t. injection of a sublethal dose of ACRO‐A (50 ng kg−1) or ACRO‐B (250 ng kg−1). Higher doses of ACRO‐A, however, could evoke allodynia dose‐dependently from 50 pg kg−1 to 500 ng kg−1 in the ACRO‐A‐treated mice. The allodynia induced by ACRO‐A (500 ng kg−1) was not inhibited by Joro spider toxin or NMDA receptor antagonists. These properties of the late allodynia induced by ACRO‐A were quite similar to those of the acute allodynia induced by ACRO‐B.

ACRO‐A could increase [Ca2+]i in the deeper laminae, rather than in the superficial laminae, of the spinal cord. This increase was not blocked by the AMPA‐preferring antagonist GYKI52466 and Joro spider toxin.

Taken together, these results demonstrate the stereospecificity of ACRO for the induction of allodynia and suggest the presence of a receptor specific to ACRO.

British Journal of Pharmacology (2004) 142, 679–688. doi:10.1038/sj.bjp.0705834
Selectins and integrins but not platelet–endothelial cell adhesion molecule‐1 regulate opioid inhibition of inflammatory pain
Authors: Machelska Halina , Brack Alexander , Mousa Shaaban A , Schopohl Julia K , Rittner Heike L , Schäfer Michael , Stein Christoph
Published: June 2004

Control of inflammatory pain can result from activation of opioid receptors on peripheral sensory nerves by opioid peptides secreted from leukocytes in response to stress (e.g. experimental swim stress or surgery). The extravasation of immunocytes to injured tissues involves rolling, adhesion and transmigration through the vessel wall, orchestrated by various adhesion molecules.

Here we evaluate the relative contribution of selectins, integrins α4 and β2, and platelet–endothelial cell adhesion molecule‐1 (PECAM‐1) to the opioid‐mediated inhibition of inflammatory pain.

We use flow cytometry, double immunofluorescence and nociceptive (paw pressure) testing in rats with unilateral hind paw inflammation induced by complete Freund's adjuvant.

In inflamed tissue, 43–58% of hematopoietic cells (CD45+) expressed opioid peptides. L‐selectin and β2 were coexpressed by 7 and 98% of opioid‐containing leukocytes, respectively. Alpha4 integrin was expressed in low levels by the majority of leukocytes. Opioid‐containing cells, vascular P‐ and E‐selectin and PECAM‐1 were simultaneously upregulated.

Swim stress produced potent opioid‐mediated antinociception in inflamed tissue, unaffected by blockade of PECAM‐1. However, blockade of L‐ and P‐selectins by fucoidin, or of α4 and β2 by monoclonal antibodies completely abolished peripheral stress‐induced antinociception. This coincided with a 40% decrease in the migration of opioid‐containing leukocytes to inflamed tissue.

These findings establish selectins and integrins α4 and β2, but not PECAM‐1, as important molecules involved in stress‐induced opioid‐mediated antinociception in inflammation. They point to a cautious use of anti‐inflammatory treatments applying anti‐selectin, anti‐α4 and anti‐β2 strategies because they may impair intrinsic pain inhibition.

British Journal of Pharmacology (2004) 142, 772–780. doi:10.1038/sj.bjp.0705837
The contribution of adrenoceptor subtype(s) in the renal vasculature of diabetic spontaneously hypertensive rats
Authors: Armenia A , Munavvar A S , Abdullah N A , Helmi A , Johns E J
Published: June 2004

Diabetes and hypertension are both associated with an increased risk of renal disease and are associated with neuropathies, which can cause defective autonomic control of major organs including the kidney. This study aimed to examine the α1‐adrenoceptor subtype(s) involved in mediating adrenergically induced renal vasoconstriction in a rat model of diabetes and hypertension.

Male spontaneously hypertensive rats (SHR), 220–280 g, were anaesthetized with sodium pentobarbitone 7‐day poststreptozotocin (55 mg kg−1 i.p.) treatment. The reductions in renal blood flow (RBF) induced by increasing frequencies of electrical renal nerve stimulation (RNS), close intrarenal bolus doses of noradrenaline (NA), phenylephrine (PE) or methoxamine were determined before and after administration of nitrendipine (Nit), 5‐methylurapidil (5‐MeU), chloroethylclonidine (CEC) and BMY 7378.

In the nondiabetic SHR group, mean arterial pressure (MAP) was 146±6 mmHg, RBF was 28.0±1.4 ml min−1 kg−1 and blood glucose was 112.3±4.7 mg dl−1, and in the diabetic SHR Group, MAP was 144±3 mmHg, RBF 26.9±1.3 ml−1 min kg−1 and blood glucose 316.2±10.5 mg dl−1. Nit, 5‐MeU and BMY 7378 blunted all the adrenergically induced renal vasoconstrictor responses in SHR and diabetic SHR by 25–35% (all P<0.05), but in diabetic rats the responses induced by RNS and NA treated with 5‐MeU were not changed. By contrast, during the administration of CEC, vasoconstrictor responses to all agonists were enhanced by 20–25% (all P<0.05) in both the SHR and diabetic SHR.

These findings suggest that α1A and α1D‐adrenoceptor subtypes contribute in mediating the adrenergically induced constriction of the renal vasculature in both the SHR and diabetic SHR. There was also an indication of a greater contribution of presynaptic adrenoceptors, that is, α1B‐, and/or α2‐subtypes.

British Journal of Pharmacology (2004) 142, 719–726. doi:10.1038/sj.bjp.0705842
Prostacyclin release and receptor activation: differential control of human pulmonary venous and arterial tone
Authors: Norel Xavier , Walch Laurence , Gascard Jean‐Pierre , Montpreville Vincent de , Brink Charles
Published: June 2004

In human pulmonary vascular preparations, precontracted arteries were more sensitive to the relaxant effect of acetylcholine (ACh) than veins (pD2 values: 7.25±0.08 (n=23) and 5.92±0.09 (n=25), respectively). Therefore, the role of prostacyclin (PGI2) was explored to examine whether this mediator may be responsible for the difference in relaxation.

In the presence of the cyclooxygenase (COX) inhibitor, indomethacin (INDO), the ACh relaxations were reduced in arteries but not in veins. On the contrary, an inhibitor (L‐NOARG) of the nitric oxide synthase blocked preferentially the relaxation in veins.

A greater release of 6‐keto‐PGF1α, the stable metabolite of PGI2, was observed in arterial preparations than in venous preparations when stimulated with either ACh or arachidonic acid (AA).

Exogenous PGI2 produced a reduced relaxant effect in the precontracted vein when compared with the artery. In the presence of the EP1‐receptor antagonist AH6809, the PGI2 relaxation of veins was similar to arteries.

In veins, AA (0.1 mM) produced a biphasic response, namely, a contraction peak (0.4–0.5 g) followed by a relaxation. These contractions in venous preparations were abolished either in the absence of endothelium or in the presence of INDO or an EP1‐receptor antagonist (AH6809, SC19220). In the arterial preparations AA induced only relaxations.

In both vascular preparations, COX‐1 but not the COX‐2 protein was detected in microsomal preparations derived from homogenized tissues or freshly isolated endothelial cells.

The differential vasorelaxations induced by ACh may be explained, in part, by a more pronounced production and release of PGI2 in human pulmonary arteries than in the veins. In addition, while PGI2 induced relaxation by activation of IP‐receptors in both types of vessels, a PGI2 constrictor effect was responsible for masking the relaxation in the veins by activation of the EP1‐receptor.

British Journal of Pharmacology (2004) 142, 788–796. doi:10.1038/sj.bjp.0705843
Redox modulation of basal and β‐adrenergically stimulated cardiac L‐type Ca2+ channel activity by phenylarsine oxide
Authors: Sims Carl , Harvey Robert D
Published: June 2004

Phenylarsine oxide (PAO) is commonly used to inhibit tyrosine phosphatase activity. However, PAO can affect a variety of different processes because of its ability to promote sulfhydryl oxidation. In the present study, we investigated the effects that PAO has on basal and β‐adrenergically stimulated L‐type Ca2+ channel activity in isolated cardiac myocytes.

Extracellular application of PAO transiently stimulated the basal L‐type Ca2+ channel activity, whereas it irreversibly inhibited protein kinase A (PKA)‐dependent regulation of channel activity by isoproterenol, forskolin and 8‐CPT‐cAMP (8‐p‐chlorophenylthioadenosine 3′,5′‐cyclic monophosphate). PAO also inhibited channel activity irreversibly stimulated in the presence of adenosine 5′‐(3‐thiotriphosphate) tetralithium salt.

Neither the stimulatory nor the inhibitory effects of PAO were affected by the tyrosine kinase inhibitor lavendustin A, suggesting that tyrosine phosphorylation is not involved.

Extracellular application of the sulfhydryl‐reducing agent dithiothreitol (DTT) antagonized both the stimulatory and inhibitory effects of PAO. Yet, following intracellular dialysis with DTT, only the inhibitory effect of PAO was antagonized.

The inhibitory effect of PAO was mimicked by intracellular, but not extracellular application of the membrane impermeant thiol oxidant 5,5′‐dithio‐bis(2‐nitrobenzoic acid).

These results suggest that the stimulatory effect of PAO results from oxidation of sulfhydryl residues at an extracelluar site and the inhibitory effect is due to redox regulation of an intracellular site that affects the response of the channel to PKA‐dependent phosphorylation. It is concluded that the redox state of the cell may play a critical role in modulating β‐adrenergic responsiveness of the L‐type Ca2+ channel in cardiac myocytes.

British Journal of Pharmacology (2004) 142, 797–807. doi:10.1038/sj.bjp.0705845
Antiapoptotic mechanisms of benidipine in the ischemic/reperfused heart
Authors: Liu Hui‐Rong , Gao Feng , Tao Ling , Yan Wen‐Li , Gao Erhe , Christopher Theodore A , Lopez Bernard L , Hu Aihua , Ma Xin L
Published: June 2004

Considerable evidence indicates that calcium plays a critical role in apoptosis. We have previously shown that benidipine, a vasodilatory calcium channel blocker, attenuates postischemia myocardial apoptosis. The present study was designed to determine the mechanisms by which benidipine exerts its antiapoptotic effect.

Adult male rats were subjected to 30 min of ischemia followed by 3 h of reperfusion. Rats were randomized to receive either vehicle or benidipine (10 μg kg−1, i.v.) 10 min before reperfusion.

Compared with rats receiving vehicle, those rats treated with benidipine had reduced postischemic myocardial apoptosis as evidenced by decreased TUNEL‐positive staining (8.4±1.2 vs 15.3±1.3%, P<0.01) and caspase‐3 activity (1.94±0.25 vs 3.43±0.29, P<0.01).

Benidipine treatment significantly reduced mitochondrial cytochrome c release and caspase‐9 activation, but had no effect on caspase‐8 activation, suggesting that benidipine exerts its antiapoptotic effect by inhibiting the mitochondrial‐mediated, but not death receptor‐mediated, apoptotic pathway.

Benidipine treatment not only increased the maximal activity of ERK1/2 at 10 min after reperfusion, but also prolonged the duration of ERK1/2 activation. Benidipine treatment had no significant effect on other apoptotic regulating molecules, such as p38 MAPK.

Taken together, our present study demonstrated for the first time the differential regulation of a calcium channel blocker. Benidipine tilted the balance between ERK1/2 and p38 MAPK toward an antiapoptotic state, decreased mitochondrial cytochrome c release, reduced caspase‐9 activation, and attenuated subsequent caspase‐3 activation and postischemic myocardial apoptosis.

British Journal of Pharmacology (2004) 142, 627–634. doi:10.1038/sj.bjp.0705847
Central blockade of nitric oxide synthesis reduces moxonidine‐induced hypotension
Authors: Moreira Thiago Santos , Takakura Ana Carolina Thomaz , Menani José V , Sato Monica Akemi , Colombari Eduardo
Published: June 2004

Nitric oxide (NO) and α2‐adrenoceptor and imidazoline agonists such as moxonidine may act centrally to inhibit sympathetic activity and decrease arterial pressure.

In the present study, we investigated the effects of pretreatment with L‐NAME (NO synthesis inhibitor), injected into the 4th ventricle (4th V) or intravenously (i.v.), on the hypotension, bradycardia and vasodilatation induced by moxonidine injected into the 4th V in normotensive rats.

Male Wistar rats with a stainless steel cannula implanted into the 4th V and anaesthetized with urethane were used. Blood flows were recorded by use of miniature pulsed Doppler flow probes implanted around the renal, superior mesenteric and low abdominal aorta.

Moxonidine (20 nmol), injected into the 4th V, reduced the mean arterial pressure (−42±3 mmHg), heart rate (−22±7 bpm) and renal (−62±15%), mesenteric (−41±8%) and hindquarter (−50±8%) vascular resistances.

Pretreatment with L‐NAME (10 nmol into the 4th V) almost abolished central moxonidine‐induced hypotension (−10±3 mmHg) and renal (−10±4%), mesenteric (−11±4%) and hindquarter (−13±6%) vascular resistance reduction, but did not affect the bradycardia (−18±8 bpm).

The results indicate that central NO mechanisms are involved in the vasodilatation and hypotension, but not in the bradycardia, induced by central moxonidine in normotensive rats.

British Journal of Pharmacology (2004) 142, 765–771. doi:10.1038/sj.bjp.0705853
In vitro elucidation of substrate specificity and bioassay of proprotein convertase 4 using intramolecularly quenched fluorogenic peptides
Authors: BASAK Sarmistha , CHRÉTIEN Michel , MBIKAY Majambu , BASAK Ajoy
Published: June 2004

The fourth member of Ca2+-dependent mammalian secretory subtilase, PC4 (proprotein convertase 4), is primarily expressed in testicular germ cell and ovarian macrophage. Its role in sperm fertilization and in early embryonic development has been demonstrated earlier through several studies, including those with PC4 null mice. A number of physiological substrates found in reproductive tissues have been postulated or identified for PC4 by various biochemical studies. These include growth factors IGF-1 (insulin-like growth factor-1) and IGF-2, hormonal polypeptide proPACAP (where PACAP stands for pituitary adenylate cyclase-activating polypeptide) and a number of surface proteins of ADAM (ADisintegrin And Metalloproteinase-like) family such as ADAM-1 (fertilin α), ADAM-2 (fertilin β), ADAM-3 (procyritestin) and ADAM-5. To provide further evidence in support of this notion and also to study the substrate specificity and bioassay of PC4, a series of intramolecularly quenched fluorogenic peptides containing the cleavage sites and several mutants were prepared. A comparative kinetic analysis and measurement of Vmax (app)/Km (app) ratio of these fluorogenic substrates against PC4 and PC7 revealed that the mutant variants of h (human) proPACAP and m (mouse) ADAM-5 derived peptides Q-PACAP141–151-mutant [Abz-141RVKNKGRR↓I150P151SY(NO2)-A-CONH2] (150A151Y replaced by PS) and Q-ADAM-5380–388-mutant [Abz-380E381PKPAR↓RP388RY(NO2)A-CONH2] (381R replaced by P) are most efficiently and selectively cleaved by PC4. Using these two and Q-IGF-263–71 peptides, we showed that the sperm extract of normal adult mice is much higher when compared with that of PC4-null mice. This suggests that these fluorogenic peptides are useful for specific bioassay of PC4 activity. In addition, kinetic studies with various peptidyl-MCA indicate that the hexapeptide Ac-KTKQLR-MCA (where MCA stands for 4-methyl coumaryl-7-amide) is most efficiently and selectively cleaved by PC4 at R↓MCA, making it another effective agent for bioassay of PC4 activity. The study concludes that the most probable sequence motif for recognition by PC4 is KXKXXR or KXXR, where X is any amino acid other than cysteine and that it prefers proline at P3, P5 and/or P2´ positions. It was also revealed that PC4 is a good candidate processing enzyme for growth factors IGF-1 and -2, neuropeptide proPACAP and several ADAM proteins such as ADAM-1, -2, -3 and -5.
Partial amino acid sequence and mRNA analysis of cytosolic pyridoxine-beta-d-glucoside hydrolase from porcine intestinal mucosa: proposed derivation from the lactase-phlorizin hydrolase gene
Authors: TSEUNG Chi-Wah , McMAHON Laura G. , VÁZQUEZ Jorge , POHL Jan , GREGORY Jesse F.
Published: June 2004

We have previously identified and purified a novel β-glucosidase, designated PNGH (pyridoxine-5´-β-d-glucoside hydrolase), from the cytosolic fraction of pig intestinal mucosal. PNGH catalyses the hydrolysis of PNG (pyridoxine-5´-β-d-glucoside), a plant derivative of vitamin B6 that exhibits partial nutritional bioavailability in humans and animals. Preliminary amino acid sequence analysis indicated regions of close similarity of PNGH to the precursor form of LPH (lactase–phlorizin hydrolase), the β-glucosidase localized to the brush-border membrane. We report in the present study amino acid sequence data for PNGH and results of Northern blot analyses, upon which we propose a common genomic origin of PNGH and LPH. Internal Edman sequencing of the PNGH band isolated by SDS/PAGE yielded data for 16 peptides, averaging 10.8 amino acids in length. These peptides from PNGH (approx. 140 kDa) were highly similar to sequences existing over most of the length of the >200 kDa precursor of rabbit LPH; however, we found no PNGH sequences that corresponded to approx. 350 amino acids between positions 463 and 812 of the LPH precursor, a region encoded by exon 7 of the LPH precursor gene (amino acids 568–784), and no sequences that corresponded to regions near the N-terminus. MS analysis of tryptic peptides yielded 25 peptides, averaging 15 amino acids, with masses that matched segments of the rabbit LPH precursor. Northern blot analysis of pig and human small intestinal polyadenylated mRNA using a non-specific LPH cDNA probe showed an expected approx. 6 kb transcript of the LPH precursor, but also an approx. 4 kb transcript that was consistent with the size predicted from the PNGH protein mass. Using a probe specific to the region encoded by exon 7, hybridization occurred only with the 6 kb transcript. Based on these observations, we propose that both PNGH and LPH enzymes have the same genomic origin, but differ in transcriptional and, possibly, post-translational processing.
Characterization of an ankyrin repeat-containing Shank2 isoform (Shank2E) in liver epithelial cells
Authors: McWILLIAMS Ryan R. , GIDEY Elizabeth , FOUASSIER Laura , WEED Scott A. , DOCTOR R. Brian
Published: June 2004

Shank proteins are a family of multidomain scaffolding proteins best known for their role in organizing the postsynaptic density region in neurons. Unlike Shank1 and Shank3, Shank2 [also known as Pro-SAP1 (proline-rich synapse-associated protein 1), CortBP1 (cortactin binding protein 1) or Spank-3] has been described as a truncated family member without an N-terminal ankyrin repeat domain. The present study utilized bioinformatics to demonstrate the presence of exons encoding ankyrin repeats in the region preceding the previously described Shank2 gene. cDNA sequencing of mRNA from epithelial cells revealed a novel spliceoform of Shank2, termed Shank2E, that encodes a predicted 200 kDa protein with six N-terminal ankyrin repeats. Shank2 mRNA from epithelial tissues was larger than transcripts in brain. Likewise, the apparent mass of Shank2 protein was larger in epithelial tissues (230 kDa) when compared with brain (165/180 kDa). Immunofluorescence and membrane fractionation found Shank2E concentrated at the apical membrane of liver epithelial cells. In cultured cholangiocytes, co-immunoprecipitation and detergent solubility studies revealed Shank2E complexed with actin and co-distributed with actin in detergent-insoluble lipid rafts. These findings indicate epithelial cells express an ankyrin repeat-containing Shank2 isoform, termed Shank2E, that is poised to co-ordinate actin-dependent events at the apical membrane.
Nucleotides and transported substrates modulate different steps of the ATPase catalytic cycle of MRP1 multidrug transporter
Authors: KERN András , SZENTPÉTERY Zsófia , LILIOM Károly , BAKOS Éva , SARKADI Balázs , VÁRADI András
Published: June 2004

The human ABC (ATP-binding cassette) transporter MRP1 (human multidrug-resistance-associated protein 1; ABCC1) is involved in the cellular extrusion of conjugated metabolites and causes multidrug resistance in tumour cells. The transport of substrate molecules by ABC proteins is energized by ATP hydrolysis, performed by two co-operating ABC units. Orthovanadate (Vi), a non-covalent inhibitor of the ABC ATPases, was found to catalyse a photo-oxidative cleavage of various ATP-binding proteins. In the present study, we have identified three Vi-cleavage sites within MRP1, and found that the cleavage reactions were variably modulated by the presence of nucleotides and by transported substrates. We concluded that Vi cleavage of MRP1 at Site I detects conformational changes due to the binding of MgATP. In contrast, Site II could be identified as part of the substrate-modulated catalytic cycle, probably containing an MRP1·MgADP·Vi transition-state-like complex. Cleavage at Site III was modulated by both the binding and hydrolysis of MgATP, in a biphasic pattern, which was also affected by the presence of transported substrates. We detected two different allosteric effects and found that they control two consecutive steps of the MRP1 ATPase catalytic cycle. Nucleotide binding to the low-affinity site accelerated the formation of the pre-hydrolytic intermediate in the other catalytic centre. Interaction of the transporter with its transported substrates stimulated a later reaction of the hydrolytic cycle, the formation of the post-hydrolytic intermediate, which could be detected in both catalytic sites by the experimental strategy used.
An inactive protein phosphatase 2A population is associated with methylesterase and can be re-activated by the phosphotyrosyl phosphatase activator
Authors: LONGIN Sari , JORDENS Jan , MARTENS Ellen , STEVENS Ilse , JANSSENS Veerle , RONDELEZ Evelien , DE BAERE Ivo , DERUA Rita , WAELKENS Etienne , GORIS Jozef , VAN HOOF Christine
Published: June 2004

We have described recently the purification and cloning of PP2A (protein phosphatase 2A) leucine carboxylmethyltransferase. We studied the purification of a PP2A-specific methylesterase that co-purifies with PP2A and found that it is tightly associated with an inactive dimeric or trimeric form of PP2A. These inactive enzyme forms could be reactivated as Ser/Thr phosphatase by PTPA (phosphotyrosyl phosphatase activator of PP2A). PTPA was described previously by our group as a protein that stimulates the in vitro phosphotyrosyl phosphatase activity of PP2A; however, PP2A-specific methyltransferase could not bring about the activation. The PTPA activation could be distinguished from the Mn2+ stimulation observed with some inactive forms of PP2A, also found associated with PME-1 (phosphatase methylesterase 1). We discuss a potential new function for PME-1 as an enzyme that stabilizes an inactivated pool of PP2A.
Ligand-dependent autophosphorylation of the insulin receptor is positively regulated by Gi-proteins
Authors: KREUZER J. , NÜRNBERG B. , KRIEGER-BRAUER H. I.
Published: June 2004

Previously, we have shown that the human insulin receptor (IR) interacts with Gi2, independent of tyrosine kinase activity and stimulates NADPH oxidase via the Gα subunit of Gi2. We have now investigated the regulatory role of Gi2-proteins in IR function. For the experiments, isolated IRs from plasma membranes of human fat cells were used. The activation of IR autophosphorylation by insulin was blocked by G-protein inactivation through GDPβS (guanosine 5´-[β-thio]disphosphate). Consistently, activation of G-proteins by micromolar concentrations of GTPγS (guanosine 5´-[γ-thio]triphosphate) induced receptor autophosphorylation 5-fold over baseline and increased insulin-induced autophosphorylation by 3-fold. In the presence of 10 µM GTPγS, insulin was active at picomolar concentrations, indicating that insulin acted via its cognate receptor. Pretreatment of the plasma membranes with pertussis toxin prevented insulin- and GTPγS-induced autophosphorylation, but did not disrupt the IR–Gi2 complex. The functional nature of the IR–Gi2 complex was made evident by insulin's ability to increase association of Gi2 with the IR. This leads to an augmentation of maximal receptor autophosphorylation induced by insulin and GTPγS. The specificity of this mechanism was further demonstrated by the use of isolated preactivated G-proteins. Addition of Gi2α and Gβγ mimicked maximal response of insulin, whereas Gαs or Gαo had no stimulatory effect. These results define a novel mechanism by which insulin signalling mediates tyrosine kinase activity and autophosphorylation of the IR through recruitment of Gi-proteins.
Expression, intracellular distribution and basis for lack of catalytic activity of the PDE4A7 isoform encoded by the human PDE4A cAMP-specific phosphodiesterase gene
Authors: JOHNSTON Lee Ann , ERDOGAN Suat , CHEUNG York Fong , SULLIVAN Michael , BARBER Rachael , LYNCH Martin J. , BAILLIE George S. , VAN HEEKE Gino , ADAMS David R. , HUSTON Elaine , HOUSLAY Miles D.
Published: June 2004

PDE4A7 is an isoform encoded by the human PDE4A cAMP-specific phosphodiesterase gene that fails to hydrolyse cAMP and whose transcripts are widely expressed. Removal of either the N- or C-terminal unique portions of PDE4A7 did not reconstitute catalytic activity, showing that they did not exert a chronic inhibitory effect. A chimera (Hyb2), formed by swapping the unique N-terminal portion of PDE4A7 with that of the active PDE4A4C form, was not catalytically active. However, one formed (Hyb1) by swapping the unique C-terminal portion of PDE4A7 with that common to all active PDE4 isoforms was catalytically active. Compared with the active PDE4A4B isoform, Hyb1 exhibited a similar Km value for cAMP and IC50 value for rolipram inhibition, but was less sensitive to inhibition by Ro-20-1724 and denbufylline, and considerably more sensitive to thermal denaturation. The unique C-terminal region of PDE4A7 was unable to support an active catalytic unit, whereas its unique N-terminal region can. The N-terminal portion of the PDE4 catalytic unit is essential for catalytic activity and can be supplied by either highly conserved sequence found in active PDE4 isoforms from all four PDE4 subfamilies or the unique N-terminal portion of PDE4A7. A discrete portion of the conserved C-terminal region in active PDE4A isoforms underpins their aberrant migration on SDS/PAGE. Unlike active PDE4A isoforms, PDE4A7 is exclusively localized to the P1 particulate fraction in cells. A region located within the C-terminal portion of active PDE4 isoforms prevents such exclusive targeting. Three functional regions in PDE4A isoforms are identified, which influence catalytic activity, subcellular targeting and conformational status.
Tissue-specific loss of fucosylated glycolipids in mice with targeted deletion of alpha(1,2)fucosyltransferase genes
Authors: IWAMORI Masao , DOMINO Steven E.
Published: June 2004

Glycolipids in epithelial tissues of the gastrointestinal tract act as receptors for enteric bacteria and are implicated in the activation of the intestinal immune system. To clarify the genes involved in the fucosylation of the major glycolipids, substrate glycolipids and fucosylated products were measured in tissues of wild-type and mutant mice lacking α(1,2)fucosyltransferase genes FUT1 or FUT2. Quantitative determination was performed by TLC-immunostaining for GA1 (Gg4Cer), FGA1 (fucosyl GA1), GM1 (II3NeuAc-Gg4Cer), FGM1 (fucosyl GM1), and Forssman glycolipids. Both FGM1 and FGA1 completely disappeared from the antrum, cecum, and colon of FUT2-null mice, but not those of FUT1-null and wild-type mice. Precursor glycolipids, GM1 and GA1, accumulated in tissues of FUT2-null mice, indicating that the FUT2-encoded enzyme preferentially participates in the fucosylation of GA1 and GM1 in these tissues. Female reproductive organs were similarly found to utilize FUT2 for the fucosylation of glycolipids FGA1 (uterus and cervix), and FGM1 (ovary), due to their absence in FUT2-null mice. In FUT1-null mice FGA1 was lost from the pancreas, but was present in wild-type and FUT2-null mice, indicating that FUT1 is essential for fucosylation of GA1 in the pancreas. Ulex europaeus agglutinin-I lectin histochemistry for α(1,2)fucose residues confirmed the absence of α(1,2)fucose residues from the apical surface of pancreatic acinar glands of FUT1-null mice. Ileum, epididymis, and testis retained specific fucosylated glycolipids, irrespective of targeted deletion of either gene, indicating either compensation for or redundancy of the α(1,2)fucosyltransferase genes in these tissues.
Ceramide sensitizes astrocytes to oxidative stress: protective role of cannabinoids
Authors: CARRACEDO Arkaitz , GEELEN Math J. H. , DIEZ María , HANADA Kentaro , GUZMÁN Manuel , VELASCO Guillermo
Published: June 2004

Cannabinoids induce apoptosis on glioma cells via stimulation of ceramide synthesis de novo, whereas they do not affect viability of primary astrocytes. In the present study, we show that incubation with Δ9-tetrahydrocannabinol did not induce accumulation of ceramide on astrocytes, although incubation of these cells in a serum-free medium (with or without cannabinoids) led to stimulation of ceramide synthesis de novo and sensitization to oxidative stress. Thus treatment with H2O2 induced apoptosis of 5-day-serum-deprived astrocytes and this effect was abrogated by pharmacological blockade of ceramide synthesis de novo. The sensitizing effect of ceramide accumulation may depend on p38 mitogen-activated protein kinase activation rather than on other ceramide targets. Finally, a protective role of cannabinoids on astrocytes is shown as a long-term incubation with cannabinoids prevented H2O2-induced loss of viability in a CB1 receptor-dependent manner. In summary, our results show that whereas challenge of glioma cells with cannabinoids induces accumulation of de novo-synthesized ceramide and apoptosis, long-term treatment of astrocytes with these compounds does not stimulate this pathway and also abrogates the sensitizing effects of ceramide accumulation.
Determination of cellular nicotinic acid-adenine dinucleotide phosphate (NAADP) levels
Authors: CHURAMANI Dev , CARREY Elizabeth A. , DICKINSON George D. , PATEL Sandip
Published: June 2004

Nicotinic acid–adenine dinucleotide phosphate (NAADP) is fast emerging as a new intracellular Ca2+-mobilizing messenger. In sea urchin egg homogenates, binding of NAADP to its receptor is not readily reversible; hence, prior incubation with low concentrations of NAADP is more effective in inhibiting subsequent binding of radiolabelled NAADP than incubating the preparation with the two ligands simultaneously [Patel, Churchill and Galione (2000) Biochem. J. 352, 725–729]. We extend this finding to show that NAADP is more effective still in inhibiting the subsequent radioligand binding at lower homogenate concentrations, an effect again quite probably due to the non-reversible nature of the receptor–ligand interaction. Enhanced sensitivity of the preparation to NAADP afforded by simple manipulation of the experimental conditions has been applied to determine low levels of NAADP in acid extracts from human red blood cells, rat hepatocytes and Escherichia coli without interference from NADP breakdown. Our improved method for the quantification of NAADP should prove useful in the further assessment of its signalling role within cells.