bluuebunny/crossref_metadata_2025 huggingface dataset:
On the selectivity of superoxide dismutase mimetics and its importance in pharmacological studies
Authors: Muscoli Carolina , Cuzzocrea Salvatore , Riley Dennis P , Zweier Jay L , Thiemermann Christoph , Wang Zhi‐Qiang , Salvemini Daniela
Published: October 2003

The list of pathophysiological conditions associated with the overproduction of superoxide expands every day. Much of the knowledge compiled on the role of this radical in disease has been gathered using the native superoxide dismutase enzyme and, more recently, by the use of superoxide dismutase knockout models or transgenic models that overexpress the various isoforms of the enzyme. Although the native enzyme has shown promising anti‐inflammatory properties in both preclinical and clinical studies, there were drawbacks and issues associated with its use as a therapeutic agent and pharmacological tool. Based on the concept that removal of superoxide modulates the course of inflammation, synthetic, low‐molecular‐weight mimetics of the superoxide dismutase enzymes that could overcome some of the limitations associated with the use of the native enzyme have been designed. In this review, we will discuss the advances made using various superoxide dismutase mimetics that led to the proposal that superoxide (and/or the product of its interaction with nitric oxide, peroxynitrite) is an important mediator of inflammation, and to the conclusion that superoxide dismutase mimetics can be utilized as therapeutic agents in diseases of various etiologies. The importance of the selectivity of such compounds in pharmacological studies will be discussed.

British Journal of Pharmacology (2003) 140, 445–460. doi:10.1038/sj.bjp.0705430
A different molecular interaction of bradykinin and the synthetic agonist FR190997 with the human B2 receptor: evidence from mutational analysis
Authors: Bellucci Francesca , Meini Stefania , Cucchi Paola , Catalani Claudio , Reichert Wolfgang , Zappitelli Sabrina , Rotondaro Luigi , Quartara Laura , Giolitti Alessandro , Maggi Carlo Alberto
Published: October 2003

Binding affinity at the [3H]‐BK binding site and activity as inositol phosphate (IP) production by the peptide bradykinin (BK) and the nonpeptide FR190997 were studied at wild‐type or point‐mutated human B2 receptors (hB2R) expressed in CHO cells.

The effect of the following mutations were analyzed: E47A (TM1), W86A and T89A (TM2), I110A, L114A and S117A (TM3), T158A, M165T and L166F (TM4), T197A and S211A (TM5), F252A, W256A and F259A (TM6), S291A, F292A, Y295A and Y295F (TM7), and the double mutation W256A/Y295F.

As the wild‐type receptor‐binding affinity of FR190997 was 40‐fold lower than BK, whereas their agonist potency was comparable, both agonists produced similar maximal effects (Emax). Mutations were evaluated as affecting the affinity and/or efficacy of FR190997 compared with BK.

Two mutations were found to impair the agonist affinity of both agonists drastically: W86A and F259A. BK agonist affinity (pEC50) was reduced by 1400‐ and 150‐fold, and that of FR190997 was reduced by 400‐ and 25‐fold, at the W86A and F259A mutant B2 receptors, respectively.

Contrary to BK, the affinity of FR190997 was selectively decreased at I110A, Y295A, and Y295F mutants (>103‐fold), and a different efficacy was measured at the Y295 mutants, FR190997 being devoid of the capability to trigger IP production at Y295A mutant.

L114A, F252A, and W256A selectively impaired the efficacy of FR190997, whereas its binding affinity was not affected. As a consequence, FR190997 behaved as a high‐affinity antagonist in blocking the IP production induced by BK.

The lack of capability of FR190997 to activate or to bind the double mutant W256A/Y295F suggests that these residues are part of the same binding site, which is also important for receptor activation by the nonpeptide ligand.

Overall, by means of mutational analysis, we indicate an hB2R recognition site for the nonpeptide agonist FR190997 (between TM3, 6, and 7), different from that of BK, and show that in the same binding crevice some mutations (L114, W256, and F252) are selectively responsible for the agonist properties of only FR190997.

British Journal of Pharmacology (2003) 140, 500–506. doi:10.1038/sj.bjp.0705454
Voltage‐dependent calcium channels are involved in neurogenic dural vasodilatation via a presynaptic transmitter release mechanism
Authors: Akerman S , Williamson D J , Goadsby P J
Published: October 2003

A missense mutation of the CACNA1A gene that encodes the α1A subunit of the voltage‐dependent P/Q‐type calcium channel has been discovered in patients suffering from familial hemiplegic migraine. This suggested that calcium channelopathies may be involved in migraine more broadly, and established the importance of genetic mechanisms in migraine.

Channelopathies share many clinical characteristics with migraine, and thus exploring calcium channel functions in the trigeminovascular system may give insights into migraine pathophysiology. It is also known that drugs blocking the P/Q‐ and N‐type calcium channels have been successful in other animal models of trigeminovascular activation and head pain.

In the present study, we used intravital microscopy to examine the effects of specific calcium channel blockers on neurogenic dural vasodilatation and calcitonin gene‐related peptide (CGRP)‐induced dilation.

The L‐type voltage‐dependent calcium channel blocker calciseptine significantly attenuated (20 μg kg−1, n=7) the dilation brought about by electrical stimulation, but did not effect CGRP‐induced dural dilation.

The P/Q‐type voltage‐dependent calcium channel blocker ω‐agatoxin‐IVA (20 μg kg−1, n=7) significantly attenuated the dilation brought about by electrical stimulation, but did not effect CGRP‐induced dural dilation.

The N‐type voltage‐dependent calcium channel blocker ω‐conotoxin‐GVIA (20 μg kg−1, n=8 and 40 μg kg−1, n=7) significantly attenuated the dilation brought about by electrical stimulation, but did not effect CGRP‐induced dural dilation.

It is thought that the P/Q‐, N‐ and L‐type calcium channels all exist presynaptically on trigeminovascular neurons, and blockade of these channels prevents CGRP release, and, therefore, dural blood vessel dilation. These data suggest that the P/Q‐, N‐ and L‐type calcium channels may be involved in trigeminovascular nociception.

British Journal of Pharmacology (2003) 140, 558–566. doi:10.1038/sj.bjp.0705456
P2X4, P2Y1 and P2Y2 receptors on rat alveolar macrophages
Authors: Bowler Jonathan W , Jayne Bailey R , Alan North R , Surprenant Annmarie
Published: October 2003

ATP receptors present on rat alveolar macrophages (NR8383 cells) were identified by recordings of membrane current, measurements of intracellular calcium, RT–PCR and immunocytochemistry.

In whole‐cell recordings with a sodium‐based internal solution, ATP evoked an inward current at −60 mV. This reversed at 0 mV. The EC50 for ATP was 18 μM in normal external solution (calcium 2 mM, magnesium 1 mM). The currents evoked by 2′,3‐O‐(4‐benzoyl)benzoyl‐ATP were about five‐fold smaller than those observed with ATP. ADP, UTP and αβ‐methylene‐ATP (αβmeATP) (up to 100 μM) had no effect. ATP‐evoked currents were potentiated up to ten‐fold by ivermectin and were unaffected by suramin (30–100 μM), pyridoxal‐phosphate‐6‐azophenyl‐(2,4‐sulphonic acid) (30–100 μM), and brilliant blue G (1 μM).

In whole‐cell recordings with a potassium‐based internal solution and low EGTA (0.01 mM), ATP evoked an inward current at −60 mV that was followed by larger outward current. ADP and UTP (1–100 μM) evoked only outward currents; these reversed polarity at the potassium equilibrium potential and were blocked by apamin (10 nM). Outward currents were also blocked by the phospholipase C inhibitor U73122 (1 μM), and they were not seen with higher intracellular EGTA (10 mM). Suramin (30 μM) blocked the outward currents evoked by ATP and UTP, but not that evoked by ADP. PPADS (10 μM) blocked the ADP‐evoked outward current without altering the ATP or UTP currents.

RT–PCR showed transcripts for P2X subunits 1, 4 and 7 (not 2, 3, 5, 6) and P2Y receptors 1, 2, 4 and 12 (not 6). Immunocytochemistry showed strong P2X4 receptor expression partly associated with the membrane, weak P2X7 staining that was not associated with the cell membrane, and no P2X1 receptor immunoreactivity.

We conclude that rat alveolar macrophages express (probably homomeric) P2X4 receptors, but find no evidence for other functional P2X subtypes. The P2Y receptors are most likely P2Y1 and P2Y2 and these couple through phospholipase C to an increase in intracellular calcium and the opening of SK type potassium channels.

British Journal of Pharmacology (2003) 140, 567–575. doi:10.1038/sj.bjp.0705459
Interactions of androgens, green tea catechins and the antiandrogen flutamide with the external glucose‐binding site of the human erythrocyte glucose transporter GLUT1
Authors: Naftalin Richard J , Afzal Iram , Cunningham Philip , Halai Mansur , Ross Clare , Salleh Naguib , Milligan Stuart R
Published: October 2003

This study investigates the effects of androgens, the antiandrogen flutamide and green tea catechins on glucose transport inhibition in human erythrocytes. These effects may relate to the antidiabetogenic effects of green tea.

Testosterone, 4‐androstene‐3,17‐dione, dehydroepiandrosterone (DHEA) and DHEA‐3‐acetate inhibit glucose exit from human erythrocytes with half‐maximal inhibitions (Ki) of 39.2±8.9, 29.6±3.7, 48.1±10.2 and 4.8±0.98 μM, respectively. The antiandrogen flutamide competitively relieves these inhibitions and of phloretin. Dehydrotestosterone has no effect on glucose transport, indicating the differences between androgen interaction with GLUT1 and human androgen receptor (hAR).

Green tea catechins also inhibit glucose exit from erythrocytes. Epicatechin 3‐gallate (ECG) has a Ki ECG of 0.14±0.01 μM, and epigallocatechin 3‐gallate (EGCG) has a Ki EGCG of 0.97±0.13 μM. Flutamide reverses these effects.

Androgen‐screening tests show that the green tea catechins do not act genomically. The high affinities of ECG and EGCG for GLUT1 indicate that this might be their physiological site of action.

There are sequence homologies between GLUT1 and the ligand‐binding domain (LBD) of hAR containing the amino‐acid triads Arg 126, Thr 30 and Asn 288, and Arg 126, Thr 30 and Asn 29, with similar 3D topology to the polar groups binding 3‐keto and 17‐β OH steroid groups in hAR LBD. These triads are appropriately sited for competitive inhibition of glucose import at the external opening of the hydrophilic pore traversing GLUT1.

British Journal of Pharmacology (2003) 140, 487–499. doi:10.1038/sj.bjp.0705460
Norcantharidin‐induced apoptosis is via the extracellular signal‐regulated kinase and c‐Jun‐NH2‐terminal kinase signaling pathways in human hepatoma HepG2 cells
Authors: Chen Yan‐Nian , Cheng Chi‐Chih , Chen Jung‐Chou , Tsauer Wei , Hsu Shih‐Lan
Published: October 2003

Norcantharidin (NCTD) is an anticancer drug routinely used against hepatoma in China. Previously, we reported that NCTD could induce mitotic arrest and apoptosis in human hepatoma HepG2 cells. However, the intracellular signaling pathways involved in NCTD‐induced apoptotic cell death are still obscure.

Caspase inhibitors were used to clarify the role of specific caspase in NCTD‐triggered apoptotic process. Results showed that activation of caspase‐9/caspase‐3 cascade is required for NCTD‐induced apoptotic death.

To decipher the upstream signals for NCTD‐induced apoptosis, we characterized the involvement of mitogen‐activated protein kinases (MAPKs), including extracellular signal‐regulated kinase (ERK), c‐Jun NH2‐terminal kinase (JNK), and p38MAPK. The role of their downstream targets, transcription factors activating protein‐1 (AP‐1), and nuclear factor κB (NF‐κB) in NCTD‐induced apoptosis was also analyzed.

Immunoblot analyses and in vitro kinase assay demonstrated that NCTD‐induced apoptosis was accompanied by the elevations of the levels of phosphorylated form and kinase activity of ERK and JNK, but not p38MAPK.

The inhibitor of ERK pathway (U0126 or PD98059) or JNK pathway (SP600125) markedly prevented kinase activation, and also greatly reduced NCTD‐induced apoptotic cell death.

Increased DNA‐binding activity of AP‐1 and NF‐κB was also observed after NCTD treatment.

Inhibition of NF‐κB activation by PDTC or inhibition of AP‐1 activation by curcumin drastically blocked NCTD‐induced cell death.

These results imply that activation of ERK and JNK, and modulation of downstream transcription factors NF‐κB and AP‐1, may be involved in NCTD‐induced apoptosis.

British Journal of Pharmacology (2003) 140, 461–470. doi:10.1038/sj.bjp.0705461
Delayed administration of interleukin‐1 receptor antagonist protects against transient cerebral ischaemia in the rat
Authors: Mulcahy Nicholas J , Ross Jerard , Rothwell Nancy J , Loddick Sarah A
Published: October 2003

The cytokine interleukin‐1 (IL‐1) has been implicated in ischaemic, excitotoxic and traumatic brain damage in rodents. The naturally occurring IL‐1 receptor antagonist (IL‐1ra) markedly reduces neuronal injury in these conditions. However, the effects of IL‐1ra on focal, transient cerebral ischaemia in the rat, which is of major clinical relevance, have not been reported.

The objectives of this study were to test the effects of IL‐1ra on cell death after temporary cerebral ischaemia, and to investigate the therapeutic time window for IL‐1ra treatment.

Ischaemia was induced by temporary (60 min) occlusion of the middle cerebral artery (MCAO) in rats, via surgical insertion (and subsequent removal) of a thread into the internal carotid artery. Damage was quantified at various times after MCAO to investigate the temporal progression of damage and establish an appropriate time to assess the effects of IL‐1ra on cell death.

Cell death was complete 18–24 h after temporary MCAO. Intracerebroventricular injection of IL‐1ra (10 μg) at the time of MCAO and 60 min later reduced the lesion volume measured 24 h (57% reduction) or 48 h (52% reduction) after MCAO. Cell death was also significantly reduced when IL‐1ra (20 μg) was administered as a single injection, 1 h (47%), 2 h (57%) or 3 h (46%) after MCAO, when compared to vehicle.

These data show that IL‐1ra markedly reduces cell death even when administration is delayed until 3 h after induction of reversible, focal cerebral ischaemia in the rat, and support our proposal that IL‐1ra may be of therapeutic benefit in stroke.

British Journal of Pharmacology (2003) 140, 471–476. doi:10.1038/sj.bjp.0705462
Ragaglitazar: a novel PPARα & PPARγ agonist with potent lipid‐lowering and insulin‐sensitizing efficacy in animal models
Authors: Chakrabarti Ranjan , Vikramadithyan Reeba K , Misra Parimal , Hiriyan Jagadheshan , Raichur Suryaprakash , Damarla Ravi K , Gershome Cynthia , Suresh Juluri , Rajagopalan Ramanujam
Published: October 2003

Ragaglitazar [(−) DRF 2725; NNC 61‐0029] is a coligand of PPARα and PPARγ.

In ob/ob mice, ragaglitazar showed significant reduction in plasma glucose, triglyceride and insulin (ED50 values <0.03, 6.1 and <0.1 mg kg−1). These effects are three‐fold better than rosiglitazone and KRP‐297. In Zucker fa/fa rats, ragaglitazar showed dose‐dependent reduction in triglyceride and insulin, hepatic triglyceride secretion and triglyceride clearance kinetics (maximum of 74, 53, 32 and 50% at 3 mg kg−1), which are better than rosiglitazone and KRP‐297.

In a high‐fat‐fed hyperlipidaemic rat model, the compound showed an ED50 of 3.95, 3.78 mg kg−1 for triglyceride and cholesterol lowering, and 0.29 mg kg−1 for HDL‐C increase. It also showed improvement in clearance of plasma triglyceride and hepatic triglyceride secretion rate. All these effects are 3–10‐fold better than fenofibrate and KRP‐297.

Ragaglitazar treatment showed significant reduction in plasma Apo B and Apo CIII levels, and increase in liver CPT1 and CAT activity and ACO mRNA. Significant increase of both liver and fat LPL activity and fat aP2 mRNA was also observed.

In a high‐fat‐fed hamster model, ragaglitazar at 1 mg kg−1 showed 83 and 61% reduction in triglyceride and total cholesterol, and also 17% reduction in fat feed‐induced body weight increase. In these hyperlipidaemic animal models, PPARγ ligands failed to show any significant efficacy. Taken together, ragaglitazar shows better insulin‐sensitizing and lipid‐lowering potential, as compared to the standard compounds.

British Journal of Pharmacology (2003) 140, 527–537. doi:10.1038/sj.bjp.0705463
Tetrahydrocannabinol‐induced neurotoxicity depends on CB1 receptor‐mediated c‐Jun N‐terminal kinase activation in cultured cortical neurons
Authors: Downer Eric J , Fogarty Marie P , Campbell Veronica A
Published: October 2003

Δ9‐Tetrahydrocannabinol (THC), the main psychoactive ingredient of marijuana, induces apoptosis in cultured cortical neurons. THC exerts its apoptotic effects in cortical neurons by binding to the CB1 cannabinoid receptor. The CB1 receptor has been shown to couple to the stress‐activated protein kinase, c‐Jun N‐terminal kinase (JNK). However, the involvement of specific JNK isoforms in the neurotoxic properties of THC remains to be established.

The present study involved treatment of rat cultured cortical neurons with THC (0.005–50 μM), and combinations of THC with the CB1 receptor antagonist, AM 251 (10 μM) and pertussis toxin (PTX; 200 ng ml−1). Antisense oligonucleotides (AS) were used to deplete neurons of JNK1 and JNK2 in order to elucidate their respective roles in THC signalling.

Here we report that THC induces the activation of JNK via the CB1 receptor and its associated G‐protein, Gi/o. Treatment of cultured cortical neurons with THC resulted in a differential timeframe of activation of the JNK1 and JNK2 isoforms.

Use of specific JNK1 and JNK2 AS identified activation of caspase‐3 and DNA fragmentation as downstream consequences of JNK1 and JNK2 activation.

The results from this study demonstrate that activation of the CB1 receptor induces JNK and caspase‐3 activation, an increase in Bax expression and DNA fragmentation. The data demonstrate that the activation of both JNK1 and JNK2 isoforms is central to the THC‐induced activation of the apoptotic pathway in cortical neurons.

British Journal of Pharmacology (2003) 140, 547–557. doi:10.1038/sj.bjp.0705464
Neurons on cannabinoids: dead or alive?
Authors: Guzmán Manuel
Published: October 2003

British Journal of Pharmacology (2003) 140, 439–440. doi:10.1038/sj.bjp.0705465
Novel data point to a broader mechanism of action of oxidized ATP: the P2X7 receptor is not the only target
Authors: Di Virgilio Francesco
Published: October 2003

Oxidized ATP (oATP) is a Schiff‐base‐forming reagent that has been used for some years as an antagonist at the P2X7 receptor (P2X7R). Preincubation of mononuclear phagocytes with this inhibitor leads to attenuation of several proinflammatory responses triggered by extracellular ATP as well as a few non‐nucleotide agonists. Novel data show that oATP reduces NFκB activation and IL‐8 release in cells lacking P2X7R, thus suggesting that some anti‐inflammatory effects of oATP may not be due to blockade of the P2X7R. This effect of oATP resembles the action of other natural or synthetic Schiff‐base‐forming reagents with immunomodulatory activity.

British Journal of Pharmacology (2003) 140, 441–443. doi:10.1038/sj.bjp.0705469
Oxidized ATP (oATP) attenuates proinflammatory signaling via P2 receptor‐independent mechanisms
Authors: Beigi Reza D , Kertesy Sylvia B , Aquilina Gretchen , Dubyak George R
Published: October 2003

Periodate‐oxidized ATP (oATP), which covalently modifies nucleotide‐binding proteins, can significantly attenuate proinflammatory signaling. Although the P2X7 nucleotide receptor (P2X7R) is irreversibly antagonized by oATP, it is unclear whether anti‐inflammatory actions of oATP are predominantly mediated via its actions on P2X7R. Here, we describe inhibitory effects of oATP on proinflammatory responses in three human cell types that lack expression of P2X7R: human umbilical vein endothelial cells (HUVEC), HEK293 cells, and 1321N1 astrocytes.

oATP decreased by 40–70% the secretion of interleukin (IL)‐8 stimulated by tumor necrosis factor‐α (TNF‐α) in all three cell types, by IL‐1β in HUVEC and 1321N1 cells, and by endotoxin in HUVEC. Attenuation of TNF‐α‐stimulated IL‐8 secretion by oATP was similar in wild‐type HEK cells or HEK cells stably expressing recombinant P2X7R.

oATP also attenuated cytokine‐stimulated expression of nuclear factor‐κB‐luciferase reporter genes expressed in HEK or 1321N1 cells, but did not affect the rapid downregulation of IκB.

oATP had no effect on uridine triphosphate‐induced activation of native P2Y2 receptors in HEK cells, but reduced the potency and efficacy of ADP as an agonist of native P2Y1 receptors. However, inhibition of P2Y1 receptors with the specific antagonist MRS2216 did not mimic the effects of oATP on TNF‐α‐stimulated IL‐8 secretion.

Although 1321N1 astrocytes lack expression of any known P2 receptor subtypes, oATP markedly inhibited ecto‐ATPase activity in these cells, resulting in a significant accumulation of extracellular ATP.

In summary, oATP can attenuate proinflammatory signaling by mechanisms independent of the expression or activation of known P2 receptor subtypes.

British Journal of Pharmacology (2003) 140, 507–519. doi:10.1038/sj.bjp.0705470
CL/RAMP2 and CL/RAMP3 produce pharmacologically distinct adrenomedullin receptors: a comparison of effects of adrenomedullin22–52, CGRP8–37 and BIBN4096BS
Authors: Hay D L , Howitt S G , Conner A C , Schindler M , Smith D M , Poyner D R
Published: October 2003

Adrenomedullin (AM) has two known receptors formed by the calcitonin receptor‐like receptor (CL) and receptor activity‐modifying protein (RAMP) 2 or 3: We report the effects of the antagonist fragments of human AM and CGRP (AM22–52 and CGRP8–37) in inhibiting AM at human (h), rat (r) and mixed species CL/RAMP2 and CL/RAMP3 receptors transiently expressed in Cos 7 cells or endogenously expressed as rCL/rRAMP2 complexes by Rat 2 and L6 cells.

AM22–52 (10 μM) antagonised AM at all CL/RAMP2 complexes (apparent pA2 values: 7.34±0.14 (hCL/hRAMP2), 7.28±0.06 (Rat 2), 7.00±0.05 (L6), 6.25±0.17 (rCL/hRAMP2)). CGRP8–37 (10 μM) resembled AM22–52 except on the rCL/hRAMP2 complex, where it did not antagonise AM (apparent pA2 values: 7.04±0.13 (hCL/hRAMP2), 6.72±0.06 (Rat2), 7.03±0.12 (L6)).

On CL/RAMP3 receptors, 10 μM CGRP8–37 was an effective antagonist at all combinations (apparent pA2 values: 6.96±0.08 (hCL/hRAMP3), 6.18±0.18 (rCL/rRAMP3), 6.48±0.20 (rCL/hRAMP3)). However, 10 μM AM22–52 only antagonised AM at the hCL/hRAMP3 receptor (apparent pA2 6.73±0.14).

BIBN4096BS (10 μM) did not antagonise AM at any of the receptors.

Where investigated (all‐rat and rat/human combinations), the agonist potency order on the CL/RAMP3 receptor was AM∼βCGRP>αCGRP.

rRAMP3 showed three apparent polymorphisms, none of which altered its coding sequence.

This study shows that on CL/RAMP complexes, AM22–52 has significant selectivity for the CL/RAMP2 combination over the CL/RAMP3 combination. On the mixed species receptor, CGRP8–37 showed the opposite selectivity. Thus, depending on the species, it is possible to discriminate pharmacologically between CL/RAMP2 and CL/RAMP3 AM receptors.

British Journal of Pharmacology (2003) 140, 477–486. doi:10.1038/sj.bjp.0705472
Electrophysiological evidence for a gradient of G protein‐gated K+ current in adult mouse atria
Authors: Lomax Alan E , Rose Robert A , Giles Wayne R
Published: October 2003

Whole cell current and voltage clamp techniques were used to examine the properties of acetylcholine‐sensitive K+ current (IKACh) in myocytes from adult mouse atrium. Superfusion of a maximal dose of carbachol (CCh; 10 μM) caused a substantial increase in K+ current in all myocytes examined.

The current–voltage (I–V) relation of maximally activated IKACh exhibited weak inward rectification. Consequently, CCh increased the amount of depolarising current necessary to evoke action potentials (APs), and APs evoked in CCh had significantly shorter durations than control APs (P<0.05).

The effects of CCh on K+ current and on AP properties were blocked by the muscarinic receptor antagonist methoctramine (1 μM). ACh (10 μM) activated a K+ current with identical properties to that activated by CCh, as did the A1 receptor agonist adenosine (100 μM).

Right atrial myocytes had significantly more IKACh than left atrial myocytes (P<0.05), regardless of whether IKACh was evoked by superfusion of muscarinic or A1 receptor agonists. IKACh current density was significantly higher in SA node myocytes than either right or left atrial myocytes.

These data identify a gradient of IKACh current density across the supraventricular structures of mouse heart. This gradient, combined with the heterogeneous distribution of parasympathetic innervation of the atria, may contribute to the proarrhythmic ability of vagal nerve stimulation to augment dispersion of atrial refractoriness.

British Journal of Pharmacology (2003) 140, 576–584. doi:10.1038/sj.bjp.0705474
Haloperidol, but not clozapine, produces dramatic catalepsy in Δ9‐THC‐treated rats: possible clinical implications
Authors: Marchese Giorgio , Casti Paola , Ruiu Stefania , Saba PierLuigi , Sanna Angela , Casu GianLuca , Pani Luca
Published: October 2003

The effect on rat catalepsy induced by Δ9‐tetrahydrocannabinol (Δ9‐THC) in association with haloperidol (HP) or clozapine (CLOZ) administration was investigated.

Δ9‐THC dose‐dependently increased HP (0.05–1 mg kg−1, s.c.)‐induced rat catalepsy, while no catalepsy was observed after CLOZ (1–20 mg kg−1, s.c.) or Δ9‐THC+CLOZ administration.

The CB1 antagonist SR141716A (0.5–5 mg kg−1, i.p.) reversed the increase mediated by Δ9‐THC on HP‐induced catalepsy.

The D2 agonist quinpirole completely reversed the catalepsy induced by both HP and HP+Δ9‐THC; however, higher doses of quinpirole were needed in the presence of Δ9‐THC.

The M1 antagonist scopolamine and α2 antagonist yohimbine were able to reduce the catalepsy induced by HP and HP+Δ9‐THC in a similar manner.

CLOZ and the 5‐HT2A/2C antagonists ritanserin, RS102221 and SB242084 were more effective in antagonizing HP than HP+Δ9‐THC‐induced catalepsy.

HP and CLOZ failed to inhibit in vitro [3H]CP‐55,940 binding, while Δ9‐THC and SR141716A did not show an appreciable affinity for the D2 receptor.

It was suggested that the different effects on rat catalepsy induced by Δ9‐THC following HP or CLOZ administration may depend on the receptor‐binding profiles of the two antipsychotics.

The preferential use of CLOZ rather than HP in the treatment of psychotic symptoms in cannabis abusers was discussed.

British Journal of Pharmacology (2003) 140, 520–526. doi:10.1038/sj.bjp.0705478
A model for the cooperative free energy transduction and kinetics of ATP hydrolysis by F
1
-ATPase
Authors: Gao Yi Qin , Yang Wei , Marcus Rudolph A. , Karplus Martin
Published: October 2003

Although the binding change mechanism of rotary catalysis by which F
1
-ATPase hydrolyzes ATP has been supported by equilibrium, kinetic, and structural observations, many questions concerning the function remain unanswered. Because of the importance of this enzyme, the search for a full understanding of its mechanism is a key problem in structural biology. Making use of the results of free energy simulations and experimental binding constant measurements, a model is developed for the free energy change during the hydrolysis cycle. This model makes possible the development of a kinetic scheme for ATP hydrolysis by F
1
-ATPase, in which the rate constants are associated with specific configurations of the β subunits. An essential new element is that the strong binding site for ADP,Pi is shown to be the β
DP
site, in contrast to the strong binding site for ATP, which is β
TP
. This result provides a rationale for the rotation of the γ subunit, which induces the cooperativity required for a tri-site binding change mechanism. The model explains a series of experimental data, including the ATP concentration dependence of the rate of hydrolysis and catalytic site occupation for both the
Escherichia coli
F
1
-ATPase (EcF
1
) and Thermophilic
Bacillus
PS3 F
1
-ATPase (TF
1
), which have different behavior.
Gating and regulation of connexin 43 (Cx43) hemichannels
Authors: Contreras Jorge E. , Sáez Juan C. , Bukauskas Feliksas F. , Bennett Michael V. L.
Published: October 2003

Connexin 43 (Cx43) nonjunctional or “unapposed” hemichannels can open under physiological or pathological conditions. We characterize hemichannels comprised of Cx43 or Cx43-EGFP (Cx43 with enhanced GFP fused to the C terminus) expressed in HeLa cells. Channel opening was induced at potentials greater than +60 mV. Open probability appeared to be very low. No comparable opening was detected in the parental, nontransfected HeLa cells. Conductance of fully open single hemichannels was ≈220 pS, which is approximately double that of Cx43 cell–cell channels. Cx43 hemichannels exhibited two types of gating: fast transitions (<1 ms) between the fully open state and a substate of ≈75 pS and slow transitions (>5 ms) between either open state and the fully closed state. Cx43-EGFP hemichannels exhibited only slow transitions (>5 ms) between closed and fully open states. These properties resemble those of the corresponding Cx43 and Cx43-EGFP cell–cell channels. Cx43 with EGFP on the N terminus (EGFP-Cx43) inserted into the surface and formed plaques but did not form hemichannels or cell–cell channels. Hemichannel blockers, 18β-glycyrrhetinic acid or La
3+
, blocked depolarization-induced currents. Uptake of ethidium bromide (
i
) was faster in Cx43 and Cx43-EGFP than parental and EGFP-Cx43 cells, (
ii
) was directly correlated with Cx43-EGFP expression, (
iii
) was reduced by hemichannel blockers, and (
iv
) occurred at the same low rate in EGFP-Cx43 and parental cells. Although hemichannel opening was not detected electrophysiologically at the resting potential, infrequent or brief opening could account for ethidium bromide uptake. Opening of Cx43 hemichannels may mediate normal signaling or be deleterious.
An enzootic transmission cycle of Lyme borreliosis spirochetes in the southeastern United States
Authors: Oliver J. H. , Lin T. , Gao L. , Clark K. L. , Banks C. W. , Durden L. A. , James A. M. , Chandler F. W.
Published: October 2003

Lyme borreliosis, or Lyme disease (LD), is a tick-borne zoonotic infection of biomedical significance, caused by
Borrelia burgdorferi sensu lato
(s.l.) spirochetes and transmitted by
Ixodes
species ticks. It usually circulates among wildlife vertebrate reservoirs and vector ticks but may infect humans, causing multisystem problems. In far western and northern North America, the host reservoirs, tick vectors, and genospecies of
Borrelia
are well known but not so in the southern U.S., where there is controversy as to the presence of “true” LD. Here we report the presence of the LD spirochete
B. burgdorferi sensu stricto
(s.s.) and
Borrelia bissettii
, three main reservoir hosts, and two enzootic tick vectors in the southeastern U.S. The two enzootic tick vectors,
Ixodes affinis
and
Ixodes minor
, rarely bite humans but are more important than the human biting “bridge” vector,
Ixodes scapularis
, in maintaining the enzootic spirochete cycle in nature. We also report extraordinary longevities and infections in the reservoir rodents
Peromyscus gossypinus
,
Sigmodon hispidus
, and
Neotoma floridana
.
Beyond the conventional description of dynamic force spectroscopy of adhesion bonds
Authors: Dudko O. K. , Filippov A. E. , Klafter J. , Urbakh M.
Published: October 2003

Dynamic force spectroscopy of single molecules is described by a model that predicts a distribution of rupture forces, the corresponding mean rupture force, and variance, which are all amenable to experimental tests. The distribution has a pronounced asymmetry, which has recently been observed experimentally. The mean rupture force follows a (ln
V
)
2/3
dependence on the pulling velocity,
V
, and differs from earlier predictions. Interestingly, at low pulling velocities, a rebinding process is obtained whose signature is an intermittent behavior of the spring force, which delays the rupture. An extension to include conformational changes of the adhesion complex is proposed, which leads to the possibility of bimodal distributions of rupture forces.
Dissecting the roles of β-catenin and cyclin D1 during mammary development and neoplasia
Authors: Rowlands Tracey M. , Pechenkina Irina V. , Hatsell Sarah J. , Pestell Richard G. , Cowin Pamela
Published: October 2003

A considerable body of circumstantial data suggests that
cyclin D1
is an attractive candidate to mediate the effects of β-catenin in mammary tissue. To test the functional significance of these correlative findings, we investigated the genetic interaction between transcriptionally active β-catenin (ΔN89β-catenin) and its target gene cyclin D1 in the mouse mammary gland during pubertal development, pregnancy, and tumorigenesis. Our data demonstrate that cyclin D1 is dispensable for the ΔN89β-catenin-stimulated initiation of alveologenesis in virgin females, for the
de novo
induction of alveoli in males, and for the formation of tumors. Indeed, lack of cyclin D1 accentuates and enhances these hyperplastic and tumorigenic ΔN89β-catenin phenotypes. Although alveologenesis is initiated by ΔN89β-catenin in a cyclin D1-independent fashion, up-regulation of cyclin D1 occurs in ΔN89β-catenin mice and its expression remains essential for the completion of alveolar development during the later stages of pregnancy. Thus, alveologenesis is a two-step process, and cyclin D1 activity during late alveologenesis cannot be replaced by the activity of other β-catenin target genes that successfully drive proliferation at earlier stages.
Single metallic nanoparticle imaging for protein detection in cells
Authors: Cognet L. , Tardin C. , Boyer D. , Choquet D. , Tamarat P. , Lounis B.
Published: October 2003

We performed a visualization of membrane proteins labeled with 10-nm gold nanoparticles in cells, using an all-optical method based on photothermal interference contrast. The high sensitivity of the method and the stability of the signals allows 3D imaging of individual nanoparticles without the drawbacks of photobleaching and blinking inherent to fluorescent markers. A simple analytical model is derived to account for the measurements of the signal amplitude and the spatial resolution. The photothermal interference contrast method provides an efficient, reproducible, and promising way to visualize low amounts of proteins in cells by optical means.
Direct observation of dipolar couplings between distant protons in weakly aligned nucleic acids
Authors: Boisbouvier Jérôme , Delaglio Frank , Bax Ad
Published: October 2003

Under conditions where macromolecules are aligned very weakly with respect to an external magnetic field, Brownian diffusion no longer averages internuclear dipole–dipole interactions to zero. The resulting residual dipolar coupling, although typically 3 orders of magnitude weaker than in a fully aligned sample, can readily be measured by solution NMR methods. To date, application of this idea has focused primarily on pairs of nuclei separated by one or two covalent bonds, where the internuclear separation is known and the measured dipolar coupling provides direct information on the orientation of the internuclear vector. A method is described that allows observation of dipolar interactions over much larger distances. By decoupling nearest-neighbor interactions, it is readily possible to observe direct dipolar interactions between protons separated by up to 12 Å. The approach is demonstrated for the DNA dodecamer d(CGCGAATTCGCG)
2
, where direct interactions are observed between protons up to three base pairs apart.
Mitochondrial positioning in fission yeast is driven by association with dynamic microtubules and mitotic spindle poles
Authors: Yaffe Michael P. , Stuurman Nico , Vale Ronald D.
Published: October 2003

Microtubules mediate mitochondrial distribution in the yeast
Schizosaccharomyces pombe
and many higher eukaryotic cells. In higher eukaryotes, kinesin motor proteins have been shown to transport mitochondria along microtubules, but the nature of the mitochondria–microtubule interactions in
S. pombe
has not been explored. By time lapse, total internal reflection fluorescence microscopy, or spinning-disk confocal microscopy, mitochondria appeared to be both tethered to ends and bound laterally along the sides of microtubules. Mitochondrial tubules extended and retracted when attached to the tips of elongating or shortening microtubules, respectively, but translocation along established microtubules was never observed. Mitochondria that were not associated with microtubules were largely immobile until they were “captured” by a growing microtubule. In mitotic cells, a portion of the mitochondria was tethered to the spindle-pole bodies and moved to the cellular ends during spindle elongation. This association may be important for organelle inheritance during cell division. Thus, in contrast to kinesin-mediated transport used by higher eukaryotes, mitochondrial motility and distribution in fission yeast are driven largely by microtubule polymerization and the elongation of the mitotic spindle.
Conserved pathways within bacteria and yeast as revealed by global protein network alignment
Authors: Kelley Brian P. , Sharan Roded , Karp Richard M. , Sittler Taylor , Root David E. , Stockwell Brent R. , Ideker Trey
Published: October 2003

We implement a strategy for aligning two protein–protein interaction networks that combines interaction topology and protein sequence similarity to identify conserved interaction pathways and complexes. Using this approach we show that the protein–protein interaction networks of two distantly related species,
Saccharomyces cerevisiae
and
Helicobacter pylori
, harbor a large complement of evolutionarily conserved pathways, and that a large number of pathways appears to have duplicated and specialized within yeast. Analysis of these findings reveals many well characterized interaction pathways as well as many unanticipated pathways, the significance of which is reinforced by their presence in the networks of both species.
Probe selection for high-density oligonucleotide arrays
Authors: Mei Rui , Hubbell Earl , Bekiranov Stefan , Mittmann Mike , Christians Fred C. , Shen Mei-Mei , Lu Gang , Fang Joy , Liu Wei-Min , Ryder Tom , Kaplan Paul , Kulp David , Webster Teresa A.
Published: October 2003

High-density oligonucleotide microarrays enable simultaneous monitoring of expression levels of tens of thousands of transcripts. For accurate detection and quantitation of transcripts in the presence of cellular mRNA, it is essential to design microarrays whose oligonucleotide probes produce hybridization intensities that accurately reflect the concentration of original mRNA. We present a model-based approach that predicts optimal probes by using sequence and empirical information. We constructed a thermodynamic model for hybridization behavior and determined the influence of empirical factors on the effective fitting parameters. We designed Affymetrix GeneChip probe arrays that contained all 25-mer probes for hundreds of human and yeast transcripts and collected data over a 4,000-fold concentration range. Multiple linear regression models were built to predict hybridization intensities of each probe at given target concentrations, and each intensity profile is summarized by a probe response metric. We selected probe sets to represent each transcript that were optimized with respect to responsiveness, independence (degree to which probe sequences are nonoverlapping), and uniqueness (lack of similarity to sequences in the expressed genomic background). We show that this approach is capable of selecting probes with high sensitivity and specificity for high-density oligonucleotide arrays.
Tumor necrosis factor α-converting enzyme mediates MUC5AC mucin expression in cultured human airway epithelial cells
Authors: Shao Matt X. G. , Ueki Iris F. , Nadel Jay A.
Published: October 2003

Ectodomain shedding of epidermal growth factor receptor (EGFR) ligands [e.g., transforming growth factor type α (TGF-α)] and EGFR phosphorylation are implicated in mucin production in airway epithelial cells. Tumor necrosis factor α-converting enzyme (TACE) is reported to cleave precursor of TGF-α, with release of soluble mature TGF-α in various epithelial tissues. We hypothesized that TACE increases the shedding of TGF-α, resulting in EGFR phosphorylation and inducing mucin production in human airway epithelial (NCI-H292) cells. To examine this hypothesis, we stimulated NCI-H292 cells with phorbol 12-myristate 13-acetate (PMA, an activator of TACE) and pathophysiologic stimuli [lipopolysaccharide (LPS) and supernatant from the Gram-negative bacteriumPseudomonas aeruginosa(PA sup)]. PMA, PA sup, and LPS increasedMUC5ACgene expression and mucin protein production, effects that were prevented by pretreatment with AG1478, a selective inhibitor of EGFR phosphorylation and by preincubation with an EGFR-neutralizing Ab or with a TGF-α-neutralizing Ab, implicating ligand (TGF-α)-dependent EGFR phosphorylation in mucin production. These stimuli induced release of soluble TGF-α, EGFR phosphorylation, and MUC5AC expression, which were blocked by the metalloprotease inhibitors tumor necrosis factor-α protease inhibitor-1 and tissue inhibitor of metalloprotease-3. We specifically knocked down the expression of metalloprotease TACE by using small interfering RNA for TACE. Knockdown of TACE inhibited PMA-, PA sup-, and LPS-induced TGF-α shedding, EGFR phosphorylation, and mucin production. From these results, we conclude that TACE plays a critical role in mucin production by airway epithelial cells by means of a TACE ligand–EGFR cascade in response to various stimuli.
Poxvirus DNA topoisomerase knockout mutant exhibits decreased infectivity associated with reduced early transcription
Authors: Da Fonseca Flavio , Moss Bernard
Published: October 2003

Vaccinia virus encodes a type I DNA topoisomerase that is highly conserved in all known poxviruses. Although the structure and catalytic activity of the enzyme were well studied, little was known about its biological function. The viral topoisomerase was thought to be essential, and roles in DNA replication, recombination, concatemer resolution, and transcription were suggested. Here, we demonstrated that the topoisomerase is not essential for replication of vaccinia virus in cultured cells, although deletion mutants formed fewer and smaller plaques on cell monolayers than wild-type virus. Purified mutant virus particles were able to bind and enter cells but exhibited reduced viral early transcription and a delay in DNA replication. Infecting with a high number of virus particles increased early mRNA and accelerated viral DNA synthesis. Processing of viral DNA concatemers into unit-length genomes was unimpaired at either a low or high multiplicity of infection. The data suggest that the primary, perhaps only, role of the poxvirus topoisomerase is to increase early transcription, which takes place within virus cores in the cytoplasm of infected cells. Because the topoisomerase functions early in infection, drugs capable of penetrating the virus core and irreversibly damaging DNA by trapping nicked DNA–topoisomerase intermediates could make potent antiviral agents.
Akt as a mediator of cell death
Authors: Luo Hongbo R. , Hattori Hidenori , Hossain Mir Ahamed , Hester Lynda , Huang Yunfei , Lee-Kwon Whaseon , Donowitz Mark , Nagata Eiichiro , Snyder Solomon H.
Published: October 2003

Protein kinase B/Akt possesses prosurvival and antiapoptotic activities and is involved in growth factor-mediated neuronal protection. In this study we establish Akt deactivation as a causal mediator of cell death. Akt deactivation occurs in multiple models of cell death includingN-methyl-d-aspartate excitotoxicity, vascular stroke, and nitric oxide (NO)- and hydrogen peroxide (H2O2)-elicited death of HeLa, PC12, and Jurkat T cells. Akt deactivation characterizes both caspase-dependent and -independent cell death. Conditions rescuing cell death, such as treatment with poly(ADP-ribose) polymerase or NO synthase inhibitors and preconditioning with sublethal concentrations ofN-methyl-d-aspartate, restore Akt activity. Infection of neurons with adenovirus expressing constitutively active Akt prevents excitotoxicity, whereas phosphatidylinositol 3-kinase inhibitors or infection with dominant negative Akt induce death of untreated neuronal cells.
Evolutionary origins of the vertebrate heart: Specification of the cardiac lineage in
Ciona intestinalis
Authors: Davidson Brad , Levine Michael
Published: October 2003

Here we exploit the extensive cell lineage information and streamlined genome of the ascidian,
Ciona intestinalis
, to investigate heart development in a basal chordate. Several cardiac genes were analyzed, including the sole
Ciona
ortholog of the
Drosophila tinman
gene, and tissue-specific enhancers were isolated for some of the genes. Conserved sequence motifs within these enhancers facilitated the isolation of a heart enhancer for the
Ciona
Hand-like gene. Altogether, these studies provide a regulatory framework for the differentiation of the cardiac mesoderm, beginning at the 110-cell stage, and extending through the fusion of cardiac progenitors during tail elongation. The cardiac lineage shares a common origin with the germ line, and zygotic transcription is first detected in the heart progenitors only after its separation from the germ line at the 64-cell stage. We propose that germ-line determinants influence the specification of the cardiac mesoderm, both by inhibiting inductive signals required for the development of noncardiac mesoderm lineages, and by providing a localized source of Wnt-5 and other signals required for heart development. We discuss the possibility that the germ line also influences the specification of the vertebrate heart.
Fruit odor discrimination and sympatric host race formation in
Rhagoletis
Authors: Linn Charles , Feder Jeffrey L. , Nojima Satoshi , Dambroski Hattie R. , Berlocher Stewart H. , Roelofs Wendell
Published: October 2003

Rhagoletis pomonella
is a model for incipient sympatric speciation (divergence without geographic isolation) by host-plant shifts. Here, we show that historically derived apple- and ancestral hawthorn-infesting host races of the fly use fruit odor as a key olfactory cue to help distinguish between their respective plants. In flight-tunnel assays and field tests, apple and hawthorn flies preferentially oriented to, and were captured with, chemical blends of their natal fruit volatiles. Because
R. pomonella
rendezvous on or near the unabscised fruit of their hosts to mate, the behavioral preference for apple vs. hawthorn fruit odor translates directly into premating reproductive isolation between the fly races. We have therefore identified a key and recently evolved (<150 years) mechanism responsible for host choice in
R. pomonella
bearing directly on sympatric host race formation and speciation.