bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of adsorption effects on measurements of ammonia, acetic acid, and methanol
Authors: Yokelson R. J. , Christian T. J. , Bertschi I. T. , Hao W. M.
Published: October 2003

We examined how adsorption and desorption of gases from inlets and a cell could affect the accuracy of closed‐cell FTIR measurements of carbon dioxide (CO2), carbon monoxide (CO), methane (CH4), nitric oxide (NO), nitrogen dioxide (NO2), methanol (CH3OH), acetic acid (CH3COOH), and ammonia (NH3). When standards were delivered to the cell through a stainless steel inlet, temporarily reduced transmission was observed for CH3OH and NH3. However, a halocarbon wax coated inlet (normally used on the system) had excellent transmission (comparable to room temperature Teflon) for both CH3OH and NH3, even at temperatures as low as 5°C. Thus the wax is valuable for coating sampling system components that cannot be fashioned from Teflon. The instrument had a delayed response (∼10–40 s) for NH3only, which was attributed to passivation of the Pyrex multipass cell. To determine sampling artifacts that could arise from the complex sample matrix presented by smoke, the closed‐cell FTIR system was intercompared with an open‐path FTIR system (which is immune to sampling artifacts) in well‐mixed smoke. A similar cell passivation delay for NH3was the only artifact found in this test. Overall, the results suggest that ∼10 s is sufficient to detect >80% of an NH3/CO ratio sampled by our fast‐flow, closed‐cell system. Longer sampling times or consecutive samples return better results. In field campaigns the closed‐cell system sampling times were normally 10 to >100 s so NH3was probably underestimated by 5–15%.
Statistical assessment of geographic areas of compliance with air quality standards
Authors: Fuentes Montserrat
Published: October 2003

A statistical method is developed to classify geographical regions according to the air quality standards for criteria pollutants. A geographic location is designated by the U.S. Environmental Protection Agency as an area of nonattainment when it does not meet the air quality standard for one of the criteria pollutants. A statistical model for air pollution is presented. This model is used to interpolate ground measurements of pollution levels at locations where there are no air quality monitors with the objective of determining the geographic areas of nonattainment. The approach presented here for interpolation takes into account that spatial patterns of air pollutants change with location. Estimates of probabilities of nonattainment are also provided, and they are used to classify the nonattainment areas by degree of severity. The approach is applied using available data from 513 sites throughout the eastern USA where ground‐level ozone is measured hourly.
Updated emissions from ocean shipping
Authors: Corbett James J. , Koehler Horst W.
Published: October 2003

Marine vessel inventories demonstrate that ship emissions cannot be neglected in assessing environmental impacts of air pollution, although significant uncertainty in these inventories remains. We address this uncertainty by employing a bottom‐up estimate of fuel consumption and vessel activity for internationally registered fleets, including cargo vessels, other commercial vessels, and military vessels. We identify model bias in previous work, which assumed internationally registered ships primarily consume international marine fuels. Updated results suggest fuel consumption is ∼289 million metric tons per year, more than twice the quantity reported as international fuel. According to our analysis, fuel used by internationally registered fleets is apparently allocated to both international and domestic fuel statistics; this implies either that ships operate along domestic routes much of the time or that marine fuel sales to these ships may be misassigned. If the former is true, then allocation of emissions to international shipping routes may underestimate near‐coastal emissions from ships. Our updated inventories increases previous ship emissions inventories for all pollutants; for example, global NOx emissions (∼6.87 Tg N) are more than doubled. This work also produces detailed sensitivity analyses of inputs to these estimates, identifying uncertainty in vessel duty‐cycle as critical to overall emissions estimates. We discuss implications for assessing ship emissions impacts.
Angiotensin AT2 receptors: cardiovascular hope or hype?
Authors: Widdop Robert E , Jones Emma S , Hannan Ruth E , Gaspari Tracey A
Published: October 2003

British Journal of Pharmacology (2003) 140, 809–824. doi:10.1038/sj.bjp.0705448
Functional role of angiotensin II AT2 receptor in modulation of AT1 receptor‐mediated contraction in rat uterine artery: involvement of bradykinin and nitric oxide
Authors: Hannan Ruth E , Davis Elizabeth A , Widdop Robert E
Published: October 2003

The aim of the present study was to explore the mechanisms underlying angiotensin II AT2 receptor modulation of AT1 receptor‐mediated vasoconstriction in the rat isolated uterine artery, since previous studies have suggested that AT2 receptors may oppose AT1 receptor‐mediated effects.

Segments of uterine artery were obtained from Sprague–Dawley rats and mounted in small vessel myographs. Concentration–response (CR) curves to angiotensin II (0.1 nM–0.1 μM) were constructed in the absence and presence of PD 123319 (AT2 antagonist; 1 μM), HOE 140 (bradykinin B2 antagonist; 0.1 μM), Nω‐nitro‐L‐arginine (NOLA) (NOS inhibitor; 30 μM), as well as combinations of these inhibitors. Contractile responses to angiotensin II were expressed as a percent of the response to a K+ depolarizing solution.

PD 123319 (1 μM) potentiated angiotensin II‐induced contractions; reflected by a significant four‐fold leftward shift of the angiotensin II CR curve.

HOE 140 (0.1 μM) significantly increased the pEC50 of the angiotensin II CR curve. The combination of HOE 140 plus PD 123319 did not produce additive potentiation.

NOLA (30 μM) significantly enhanced sensitivity to angiotensin II, seen as a five‐fold leftward shift of the curve, and an augmented maximum contractile response. Combinations of PD 123319 (1 μM) plus NOLA, and of HOE 140 (0.1 μM) plus NOLA, both induced a similar magnitude of potentiation.

Cyclic GMP measurements confirmed angiotensin II‐induced activation of the nitric oxide (NO) pathway.

In conclusion, AT2 receptor‐mediated inhibition of angiotensin II‐induced contraction of the rat uterine artery involves NO production; a component of which occurs through a bradykinin B2 receptor pathway.

British Journal of Pharmacology (2003) 140, 987–995. doi:10.1038/sj.bjp.0705484
Rho‐kinase inhibitor inhibits both myosin phosphorylation‐dependent and ‐independent enhancement of myofilament Ca2+ sensitivity in the bovine middle cerebral artery
Authors: Maeda Yoshihisa , Hirano Katsuya , Nishimura Junji , Sasaki Tomio , Kanaide Hideo
Published: October 2003

The role of Rho kinase in Ca2+ sensitization of the contractile apparatus in smooth muscle was investigated in the bovine middle cerebral artery.

U46619, a thromboxane A2 analog, induced a greater sustained contraction with a smaller [Ca2+]i elevation than that seen with 118 mM K+. The level of myosin light chain (MLC) phosphorylation obtained in the initial phase of the contraction was higher than that seen with 118 mM K+; thereafter, it gradually declined to a comparable level in the late phase.

During the steady state of the U46619‐induced contraction, Y27632 (10 μM), a Rho‐kinase inhibitor, partially inhibited [Ca2+]i, although it substantially inhibited tension and MLC phosphorylation. Wortmannin (10 μM), an MLC kinase inhibitor, had no significant effect on [Ca2+]i, but it completely inhibited MLC phosphorylation and partially inhibited tension. The wortmannin‐resistant tension development was thus not associated with MLC phosphorylation, and this component was completely inhibited by Y27632.

In conclusion, U46619 enhanced Ca2+ sensitivity in a manner both dependent and independent of MLC phosphorylation in the bovine middle cerebral artery. Both mechanisms of Ca2+ sensitization can be inhibited by the Rho‐kinase inhibitor.

British Journal of Pharmacology (2003) 140, 871–880. doi:10.1038/sj.bjp.0705487
Riluzole inhibits spontaneous Ca2+ signaling in neuroendocrine cells by activation of K+ channels and inhibition of Na+ channels
Authors: Beltran‐Parrazal Luis , Charles Andrew
Published: October 2003

The neuroprotective drug riluzole has multiple effects on cellular signaling. We found that riluzole rapidly and reversibly inhibited spontaneous Ca2+ oscillations in both immortalized GnRH‐secreting hypothalamic neurons (GT1 cells) and in the prolactin and growth‐hormone‐secreting GH3 cell line. At lower concentrations (100 nM–5 μM), riluzole reduced the amplitude and frequency of spontaneous Ca2+ oscillations, whereas at higher concentrations it abolished spontaneous Ca2+ signaling.

Whole‐cell current clamp recordings in GH3 cells revealed that riluzole decreased the action potential frequency, amplitude, and duration.

Riluzole inhibited voltage‐gated Na+ currents, increased iberiotoxin‐sensitive voltage‐gated K+ currents, and had no effect on voltage‐gated Ca2+ currents in GH3 cells. Riluzole also inhibited voltage‐gated Na+ currents and increased voltage‐gated K+ channels in GT1 cells.

The inhibitory effects of riluzole on Ca2+ signaling were blocked by pretreatment with iberiotoxin in GH3 cells, but only partially reduced by iberiotoxin in GT1 cells.

These results indicate that riluzole inhibits Ca2+ signaling primarily by activation of K+ channels in GH3 cells, and also by inhibition of Na+ channels in GT1 cells. Riluzole's inhibition of spontaneous excitability and Ca2+ signaling may be involved in its multiple effects on cellular function in the nervous system.

British Journal of Pharmacology (2003) 140, 881–888. doi:10.1038/sj.bjp.0705491
Effects of TRA‐418, a novel TP‐receptor antagonist, and IP‐receptor agonist, on human platelet activation and aggregation
Authors: Miyamoto Mitsuko , Yamada Naohiro , Ikezawa Shiho , Ohno Michihiro , Otake Atsushi , Umemura Kazuo , Matsushita Teruo
Published: October 2003

{4‐[2‐(1,1‐Diphenylethylsulfanyl)‐ethyl]‐3,4‐dihydro‐2H‐benzo[1,4]oxazin‐8‐yloxy}‐acetic acid N‐Methyl‐D‐glucamine salt (TRA‐418) has both thromboxane A2 (TP)‐receptor antagonist and prostacyclin (IP)‐receptor agonist properties. The present study examined the advantageous effects of TRA‐418 based on the dual activities, over an agent having either activity alone and also the difference in the effects of TRA‐418 and a glycoprotein αIIb/β3 integrin (GPIIb/IIIa) inhibitor.

TRA‐418 inhibited platelet GPIIb/IIIa activation as well as P‐selectin expression induced by adenosine 5′‐diphosphate, thrombin receptor agonist peptide 1–6 (Ser‐Phe‐Leu‐Leu‐Arg‐Asn‐NH2), and U‐46619 in the presence of epinephrine (U‐46619+ epinephrine). TRA‐418 also inhibited platelet aggregation induced by those platelet‐stimulants in Ca2+ chelating anticoagulant, citrate and in nonchelating anticoagulant, D‐phenylalanyl‐L‐prolyl‐L‐arginyl‐chloromethyl ketone (PPACK).

The TP‐receptor antagonist SQ‐29548 inhibited only U‐46619+epinephrine‐induced GPIIb/IIIa activation, P‐selectin expression, and platelet aggregation.

The IP‐receptor agonist beraprost sodium inhibited platelet activation. Beraprost also inhibited platelet aggregation induced by platelet stimulants we tested in citrate and in PPACK.

The GPIIb/IIIa inhibitor abciximab blocked GPIIb/IIIa activation and platelet aggregation. However, abciximab showed slight inhibitory effects on P‐selectin expression.

TRA‐418 is more advantageous as an antiplatelet agent than TP‐receptor antagonists or IP‐receptor agonists separately used. TRA‐418 showed a different inhibitory profile from abciximab in the effects on P‐selectin expression.

British Journal of Pharmacology (2003) 140, 889–894. doi:10.1038/sj.bjp.0705499
Activation and proliferation signals in primary human T lymphocytes inhibited by ergosterol peroxide isolated from Cordyceps cicadae
Authors: Kuo Y C , Weng S C , Chou C J , Chang T T , Tsai W J
Published: October 2003

Effects of ergosterol peroxide (C28H44O3; Cpd 6A) from Cordyceps cicadae on phytohemagglutinin (PHA)‐stimulated cell proliferation were studied in primary human T cells.

The results showed that Cpd 6A suppressed T‐cell proliferation for about 24 h after stimulation with PHA. Cell cycle analysis indicated that Cpd 6A arrested the cell cycle progression of activated T cells from the G1 transition to the S phase.

To localize the point in the cell cycle where arrest occurred, a set of key regulatory events leading to the G1/S boundary, including the expression of cyclins D2, E, A1, and B1, interleukin (IL)‐2, IL‐4, interferon‐γ (IFN‐γ), and activating protein‐1 (AP‐1), was examined.

Cpd 6A suppressed, in activated T lymphocytes, the production and mRNA expression of cyclin E, IL‐2, IL‐4, IL‐10, and IFN‐γ in a dose‐dependent manner.

Expression of AP‐1 proteins, consisting of c‐Fos and c‐Jun, in activated T lymphocytes was decreased by Cpd 6A. The kinetic study indicated that the inhibitory effects of Cpd 6A on IL‐2 mRNA expressed in T cells might be related to blocking c‐Fos protein synthesis. T‐cell proliferation after Cpd 6A treatment was partially restored by addition of IL‐2, IL‐4, and IFN‐γ.

These suppressant effects of Cpd 6A on T‐cell proliferation, activated by PHA, appeared to be mediated, at least in part, through the inhibition of early gene transcripts, especially those of cyclin E, IFN‐γ, IL‐2, and IL‐4, and by arresting cell cycle progression in the cells.

British Journal of Pharmacology (2003) 140, 895–906. doi:10.1038/sj.bjp.0705500
Acute effects of dronedarone on both components of the cardiac delayed rectifier K+ current, HERG and KvLQT1/minK potassium channels
Authors: Thomas Dierk , Kathöfer Sven , Zhang Wei , Wu Kezhong , Wimmer Anna‐Britt , Zitron Edgar , Kreye Volker A W , Katus Hugo A , Schoels Wolfgang , Karle Christoph A , Kiehn Johann
Published: October 2003

Dronedarone is a noniodinated benzofuran derivative that has been synthesized to overcome the limiting iodine‐associated adverse effects of the potent antiarrhythmic drug amiodarone. In this study, the acute electrophysiological effects of dronedarone on repolarizing potassium channels were investigated to determine the class III antiarrhythmic action of this compound. HERG and KvLQT1/minK potassium channels conduct the delayed rectifier potassium current IK in human heart, being a primary target for class III antiarrhythmic therapy.

HERG and KvLQT1/minK were expressed heterologously in Xenopus laevis oocytes, and the respective potassium currents were recorded using the two‐microelectrode voltage‐clamp technique.

Dronedarone blocked HERG channels with an IC50 value of 9.2 μM and a maximum tail current reduction of 85.2%.

HERG channels were blocked in the closed, open, and inactivated states. The half‐maximal activation voltage was shifted by −6.1 mV, and HERG current block by dronedarone was voltage‐dependent, but not use‐dependent.

Dronedarone exhibited a weaker block of KvLQT1/minK currents (33.2% at 100 μM drug concentration), without causing significant changes in the corresponding current–voltage relationships.

In conclusion, these data demonstrate that dronedarone is an antagonist of cloned HERG potassium channels, with additional inhibitory effects on KvLQT1/minK currents at higher drug concentrations, providing a molecular mechanism for the class III antiarrhythmic action of the drug.

British Journal of Pharmacology (2003) 140, 996–1002. doi:10.1038/sj.bjp.0705502
Rivastigmine blocks voltage‐activated K+ currents in dissociated rat hippocampal neurons
Authors: Pan Yaping , Xu Xianghua , Wang Xiaoliang
Published: October 2003

Rivastigmine is an acetylcholinesterase inhibitor used in Alzheimer's disease therapy. In the present study, we investigated the effects of rivastigmine on the transient outward K+ current (IK(A)) and the delayed rectifier K+ current (IK(DR)) in acutely dissociated rat hippocampal pyramidal neurons using the whole‐cell patch‐clamp technique.

Rivastigmine inhibited the amplitudes of IK(A) and IK(DR) in a reversible and concentration‐dependent manner. At a concentration of 100 μM, rivastigmine inhibited IK(A) and IK(DR), recorded when the cells were depolarized from −50 to +40 mV, by 65.9 (P<0.01) and 67.3% (P<0.01), respectively. The IC50 values for IK(A) and IK(DR) were 3.8 and 1.7 μM, respectively.

The decay time constant of IK(A), recorded following a test pulse to +40 mV, was prolonged reversibly by rivastigmine at concentrations of 10 and 100 μM (both P<0.05).

Rivastigmine affected the voltage dependence of IK(A) and IK(DR). At a concentration of 10 μM, it shifted the steady‐state inactivation curve of IK(A) towards more negative potentials by −11 mV (P<0.05), but had no effect on the steady‐state activation curve or the recovery from inactivation. Regarding the kinetic properties of IK(DR), 10 μM rivastigmine shifted the steady‐state activation and inactivation curves towards more negative potentials by −10 (P<0.05) and −27 mV (P<0.01), respectively.

Our findings that rivastigmine inhibits IK(A) and IK(DR) in rat hippocampal pyramidal neurons suggest that this agent has other pharmacological actions besides its antiacetylcholinesterase activity.

British Journal of Pharmacology (2003) 140, 907–912. doi:10.1038/sj.bjp.0705503
Importance of ERK activation in behavioral and biochemical effects induced by MDMA in mice
Authors: Salzmann Julie , Marie‐claire Cynthia , Guen Stéphanie Le , Roques Bernard P , Noble Florence
Published: October 2003

Little is known about the cellular effects induced by 3,4‐methylenedioxymethamphetamine (MDMA, ecstasy), although changes in gene expression have been observed following treatments with other psychostimulants. Thus, the aim of this study was to investigate in mice, the relationships between the ras‐dependent protein kinase ERK and MDMA‐induced reinforcement using the conditioned place preference (CPP) and locomotor activity measurements. This was completed using real‐time quantitative PCR method by a study of immediate early‐genes (IEGs) transcription known to be involved in neuronal plasticity.

A significant CPP was observed after repeated MDMA treatment in CD‐1 mice at a dose of 9 mg kg−1 i.p. but not at 3 and 6 mg kg−1. This rewarding effect was abolished by the selective inhibitor of ERK activation, SL327 (50 mg kg−1; i.p.). Similar results were obtained on MDMA‐induced locomotor activity, clearly suggesting a role of ERK pathway in these behavioral responses.

Following acute i.p. injection, MDMA induced a strong c‐fos transcription in brain structures, such as caudate putamen, nucleus accumbens and hippocampus, whereas egr‐1 and egr‐3 transcripts were only increased in the caudate putamen. MDMA‐induced IEGs transcription was selectively suppressed by SL327 in the caudate putamen, suggesting a role for other signaling pathways in regulation of IEGs transcription in the other brain structures. In agreement with these results, MDMA‐induced c‐fos protein expression was blocked by SL327 in the caudate putamen.

This study confirms and extends to mice the reported role of ERK pathway in the development of addiction‐like properties of MDMA. This could facilitate studies about the molecular mechanism of this process by using mutant mice.

British Journal of Pharmacology (2003) 140, 831–838. doi:10.1038/sj.bjp.0705506
In vivo characterization of 5‐HT1A receptor‐mediated gastric relaxation in conscious dogs
Authors: Janssen P , Prins N H , Moreaux B , Meulemans A L , Lefebvre R A
Published: October 2003

Accumulating data have been published emphasizing the important role of 5‐hydroxytryptamine (5‐HT) receptors in proximal stomach relaxation. However, a proper in vivo characterization of 5‐HT receptors mediating gastric relaxation is still missing. In the current study, we focus on the in vivo characterization of 5‐HT1A receptors mediating relaxation of the proximal stomach in conscious dogs.

Beagle dogs were equipped with a gastric fistula. In the conscious state, volume changes within an intragastric bag were measured at constant pressure by means of a barostat. Results are presented as the maximum volume increase after treatment (mean±s.e.m.). All drugs were injected intravenously.

The 5‐HT1A receptor agonist flesinoxan (10, 50, 100 and 150 μg kg−1) induced a dose‐dependent relaxation of the canine proximal stomach (50±10, 230±51, 290±38 and 275±33 ml, respectively; n=9–11). The selective 5‐HT1A receptor antagonist WAY‐100635 dose‐dependently inhibited the flesinoxan‐induced relaxation. NG‐nitro‐L‐arginine methyl ester did not affect this relaxation, suggesting that nitrergic nerves are not involved.

After supradiaphragmatic vagotomy, the baseline of the intragastric volume was larger compared to that before vagotomy (317±50 vs 142±28 ml, respectively; n=5). Compensation for this by either reduction of the intraballoon pressure or infusion of a contractile dose of bethanechol did not establish a condition in which flesinoxan was able to relax the stomach. In contrast, nitroprusside induced a significant gastric relaxation when tone was increased by bethanechol.

It is concluded that flesinoxan induces proximal gastric relaxation in conscious dogs via 5‐HT1A receptors. The response is mediated through a vagal pathway without involvement of nitrergic nerves.

British Journal of Pharmacology (2003) 140, 913–920. doi:10.1038/sj.bjp.0705507
Theophylline attenuates Ca2+ sensitivity and modulates BK channels in porcine tracheal smooth muscle
Authors: Ise Shinji , Nishimura Junji , Hirano Katsuya , Hara Nobuyuki , Kanaide Hideo
Published: October 2003

Theophylline, a nonselective phosphodiesterase inhibitor, has long been regarded as a major bronchodilator in the treatment of human asthma. Using front‐surface fluorometry with fura‐2 and α‐toxin permeabilization, the effects of theophylline on intracellular Ca2+ concentration ([Ca2+]i), tension development and Ca2+ sensitivity of the contractile apparatus were investigated in porcine tracheal smooth muscle strips.

Application of theophylline induced a relaxation without a significant decrease in [Ca2+]i when strips were precontracted by 40 mM K+ depolarization, while theophylline significantly decreased both [Ca2+]i and tension induced by carbachol.

The effects of theophylline on the increases in [Ca2+]i and tension induced by carbachol were significantly inhibited by iberiotoxin, an inhibitor of large‐conductance Ca2+‐activated K+ channels.

In the absence of extracellular Ca2+, theophylline significantly attenuated carbachol‐induced transient increases in tension development, while it did not affect carbachol‐induced transient increase in [Ca2+]i.

The [Ca2+]i–force relationship, which was determined by cumulative applications of extracellular Ca2+ (0–5 mM) during 40 mM K+ depolarization, was significantly shifted to the right by theophylline.

In α‐toxin permeabilized strips, theophylline significantly increased the EC50 value of [Ca2+]i for contraction and enhanced the effect of cAMP, but not of cGMP.

These results indicate that theophylline induces relaxation of the porcine tracheal smooth muscle through an activation of BK channels, and a resultant decrease in [Ca2+]i and an attenuation of Ca2+ sensitivity, presumably through the action of cAMP.

British Journal of Pharmacology (2003) 140, 939–947. doi:10.1038/sj.bjp.0705508
Inhibition by ethanol of NMDA‐induced responses and acute tolerance to the inhibition in rat sympathetic preganglionic neurons in vitro and in vivo
Authors: Lin Hsun Hsun , Hsieh Wei‐Kung , Shiu Jing‐Yi , Chiu Ted H , Lai Chih‐Chia
Published: October 2003

N‐methyl‐D‐aspartate (NMDA) receptors have been demonstrated to be a pivotal target for ethanol action. The present study examined the actions of acute ethanol exposure on NMDA‐induced responses and the acute tolerance to ethanol actions in rat sympathetic preganglionic neurons (SPNs) in vitro and in vivo.

NMDA (50 μM) applied every 5 min induced reproducible membrane depolarizations of SPNs in neonatal spinal cord slice preparations. Ethanol (50 – 100 mM) applied by superfusion for 15 min caused a sustained decrease in NMDA‐induced depolarizations in a dose‐dependent and reversible manner. When the superfusion time of ethanol (100 mM) was increased to 50 min, NMDA‐induced depolarizations were attenuated initially but a gradual recovery was seen in ∼40% of SPNs tested.

Repeated injections of NMDA (2 nmol) intrathecally at 30 min interval caused reproducible increases in mean arterial pressure (MAP) in urethane‐anesthetized rats. Intravenous injections of ethanol (0.16 or 0.32 g, 1 ml) inhibited NMDA‐induced pressor effects in a blood concentration‐dependent manner. The inhibition by ethanol of NMDA‐induced pressor effects was reduced over time during continuous infusion of ethanol or on the second injection 3.5 h after prior injection of a higher dose of ethanol.

Ethanol, at concentrations significantly inhibited NMDA‐induced responses, had no significant effects on α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid‐induced responses. The study demonstrated the selective inhibition by ethanol of NMDA‐induced responses and the development of acute tolerance to the inhibitory effects in SPNs both in vitro and in vivo. These effects may play important roles in the ethanol regulation of cardiovascular function.

British Journal of Pharmacology (2003) 140, 955–963. doi:10.1038/sj.bjp.0705512
Inhibition of interleukin‐12 expression by α‐thrombin in human peripheral blood mononuclear cells: a potential mechanism for modulating Th1/Th2 responses
Authors: Naldini A , Aarden L , Pucci A , Bernini C , Carraro F
Published: October 2003

In addition to its central role in blood coagulation and hemostasis, human α‐thrombin is a powerful regulator of inflammatory responses and is known to affect cell‐mediated immunity. Interleukin (IL)‐12 is a strong promoter of the development of Th1‐type lymphocytes and its downregulation implies a positive feedback mechanism for development of Th2 responses. We have previously shown that thrombin enhances the release of IL‐6, a Th2‐related cytokine, in human peripheral blood mononuclear cells (PBMC).

Here we show that thrombin downregulates IL‐12 production at both protein and mRNA levels in human PBMC. The inhibition of IL‐12 production was accompanied by an enhanced release of IL‐10, which inhibits Th1‐related processes and promotes Th2‐type responses.

The use of proteolytically inactive thrombin and of the specific thrombin receptor agonist peptide, SFLLRN, reveals that this downregulation is thrombin‐specific and requires thrombin proteolytic activity.

In addition, activation of coagulation inhibits IL‐12 production in whole blood cultures, confirming the tight relationship between the coagulation pathway, where thrombin is a key enzyme, and inflammation.

Decreased IL‐12 production appears to be related also to IL‐10 production, since the addition of an anti‐IL‐10 monoclonal antibody to thrombin‐treated PBMC resulted in a partial restoration of IL‐12 production.

In conclusion, the observation that thrombin significantly affects the production of IL‐12, as well as of IL‐10, implies a concerted role orchestrated by thrombin in PBMC that could be crucial to effective immunity and inflammation.

British Journal of Pharmacology (2003) 140, 980–986. doi:10.1038/sj.bjp.0705514
Impaired host defense to Klebsiella pneumoniae infection in mice treated with the PDE4 inhibitor rolipram
Authors: Soares A C , Souza D G , Pinho V , Vieira A T , Barsante M M , Nicoli J R , Teixeira M
Published: October 2003

The increase in levels of cAMP in leukocytes by selective inhibitors of PDE4 may result in reduction of inflammation, and may be useful in the treatment of pulmonary inflammatory disorders in humans. Here, we have assessed whether oral treatment with the prototype PDE4 inhibitor, rolipram, interfered with the antibacterial host response following pulmonary infection of mice with Klebsiella pneumoniae.

K. pneumoniae infection induced a marked increase in the recruitment of neutrophils to the lungs and the production of proinflammatory cytokines and chemokines, including tumor necrosis factor‐α (TNF‐α) and keratinocyte‐derived chemokine (KC), in bronchoalveolar (BAL) fluid and lung tissue. There were also detectable amounts of interleukin‐10 (IL‐10) and significant lethality.

Treatment with rolipram (3–30 mg kg−1) was associated with earlier lethality and significant inhibition of the TNF‐α production. This was associated with enhanced production of IL‐10 in lung tissue of rolipram‐treated animals. Rolipram treatment did not affect KC expression and the recruitment of neutrophils in the lung tissue.

Over 70% of neutrophils that migrated into the BAL fluid following K. pneumoniae infection ingested bacteria. Treatment with rolipram inhibited the percentage of neutrophils undergoing phagocytosis of K. pneumoniae in a dose‐dependent manner. Maximal inhibition (62%) occurred at doses equal to or greater than 10 mg kg−1.

Thus, treatment of mice with the PDE4 inhibitor rolipram is accompanied by earlier lethality, enhanced bacterial load and decreased capacity of the responding host to produce TNF‐α and of neutrophils to phagocytose bacteria. It will be important to investigate whether the shown ability of PDE4 inhibitors to inhibit neutrophil phagocytosis and control experimental bacterial infection will translate into an inhibition of the ability of neutrophils to deal with infectious microorganisms in the clinical setting.

British Journal of Pharmacology (2003) 140, 855–862. doi:10.1038/sj.bjp.0705517
Block of cardiac delayed‐rectifier and inward‐rectifier K+ currents by nisoldipine
Authors: Missan Sergey , Zhabyeyev Pavel , Dyachok Oksana , Jones Stephen E , McDonald Terence F
Published: October 2003

The objective of this study was to determine the concentration‐dependent effects of nisoldipine, a dihydropyridine Ca2+ channel blocker, on K+ currents in guinea‐pig ventricular myocytes.

Myocytes in the conventional whole‐cell configuration were bathed in normal Tyrode's solution or K+‐free Tyrode's solution for the measurement of the effects of 0.01–100 μM nisoldipine on rapidly activating delayed‐rectifier K+ current (IKr), slowly activating delayed‐rectifier K+ current (IKs), inwardly rectifying K+ current (IK1), and reference L‐type Ca2+ current (ICa,L).

Nisoldipine inhibited IKr with an IC50 of 23 μM, and IKs with an IC50 of 40 μM. The drug also had weak inhibitory effects on inward‐ and outward‐directed IK1; the IC50 determined for outward‐directed current was 80 μM.

Investigation of nisoldipine action on IKs showed that inhibition occurred in the absence of previous pulsing, and with little change in the time courses of activation and deactivation. However, the drug‐induced inhibition was significantly weaker at +30 mV than at +10 mV.

We estimate that nisoldipine is about 30 times less selective for delayed‐rectifier K+ channels than for L‐type Ca2+ channels in fully polarised guinea‐pig ventricular myocytes, and several orders less selective in partially depolarised myocytes.

British Journal of Pharmacology (2003) 140, 863–870. doi:10.1038/sj.bjp.0705518
Identification and characterisation of GPR100 as a novel human G‐protein‐coupled bradykinin receptor
Authors: Boels Katrin , Schaller H Chica
Published: October 2003

G‐protein‐coupled receptor 100 (GPR100) was discovered by searching the human genome database for novel G‐protein‐coupled peptide receptors.

Full‐length GPR100 was amplified from a cDNA library of the neuroendocrine cell line BON, which is derived from a human pancreas carcinoid. The open‐reading frame, present on a single exon, coded for a protein of 374 amino acids with highest sequence identity (43%) to the human orphan somatostatin‐ and angiotensin‐like peptide receptor. The analysis of chromosomal localisation mapped the GPR100 gene to chromosome 1q21.2–q21.3.

The stable expression of GPR100 in Chinese hamster ovary cells together with aequorin as calcium sensor and the promiscuous G‐protein subunit α16 as signal transducer revealed bradykinin and kallidin as effectors to elicit a calcium response. Dose–response curves yielded EC50 values for both ligands in the low nanomolar range, while the respective analogues without arginine at the carboxy‐terminus were inactive. Calcium mobilisation was inhibited by the phospholipase C blocker U73122, but not by pertussis toxin, suggesting the involvement of the G‐protein subunit αq and not αi or αo in signal transduction.

In line with the main function of kinins as peripheral hormones, we found that GPR100 was expressed predominantly in tissues like pancreas, heart, skeletal muscle, salivary gland, bladder, kidney, liver, placenta, stomach, jejunum, thyroid gland, ovary, and bone marrow, but smaller amounts were also detected in the brain and in cell lines derived from tumours of various origins.

British Journal of Pharmacology (2003) 140, 932–938. doi:10.1038/sj.bjp.0705521
Corrigendum to Halogenation of a capsaicin analogue leads to novel vanilloid TRPVI receptor antagonists
Authors: Appendino Giovanni , Harrison Selena , Petrocellis Luciano De , Daddario Nives , Bianchi Federica , Moriello Aniello Schiano , Trevisani Marcello , Benvenuti Francesca , Geppetti Pierangelo , Marzo Vincenzo Di
Published: October 2003

British Journal of Pharmacology (2003) 140, 1008. doi:10.1038/sj.bjp.0705565
Screening for glycosylation changes on recombinant human IgG using lectin methods
Authors: Fotinopoulou Angeliki , Meyers Timothy , Varley Paul , Turner Graham
Published: October 2003

Two lectin‐binding methods were investigated as possible ways of monitoring the glycosylation of human monoclonal antibodies during their development and production. Carbohydrate composition was assessed in various preparations that were produced in different host cell types, cell sublines or batches of the same cells. The lectin binding was measured with ELISA and surface plasmon resonance (SPR). For comparative purposes, the monosaccharide content of many of the preparations was also measured by high‐pressure anion exchange chromatography (HPAEC)/pulsed amperometric detection (PAD). Both lectin methods detected modifications in glycosylation when antibodies were produced in different ways; SPR was more sensitive than ELISA for some lectins and vice versa. Generally, the lectin results agreed with those obtained by the monosaccharide analysis; however, the former were much better for assessing N‐acetylneuraminic acid changes. The latter were impossible to assess by HPAEC/PAD because of their low levels. The lectin‐based methods also had the advantages that they were quicker to perform and required less expertise and could quickly identify structures that monosaccharide analysis might miss. It is suggested that, in the development of therapeutic proteins, monosaccharide analysis and/or oligosaccharide profiling is initially performed but later routine batches of the glycoprotein are screened with a lectin method. Of the two lectin methods used, SPR is much quicker when performing a screen, whereas ELISA is particularly useful for comparing a particular carbohydrate feature on different samples of the same glycoprotein.
Comparison of cellular stress levels and green‐fluorescent‐protein expression in several Escherichia coli strains
Authors: Hyun Seo Jeong , Gyun Kang Dong , Joon Cha Hyung
Published: October 2003

Constructs comprising stress‐gene promoter elements from rpoH (Sigma 32), clpB or dnaK linked to a green‐fluorescent‐protein (GFP) expression vector were previously used as non‐invasive ‘stress probes’ in Escherichia coli . We compared cellular stress responses in four E. coli strains: production hosts JM105 and BL21, and cloning hosts HB101 and TOP10. When GFP was also used as a model for foreign protein production, we generally observed that the level of expression was inversely proportional to the level of cellular stress. JM105 showed the highest cellular stress level and very low GFP expression, while BL21 exhibited the lowest cellular stress level and the highest GFP expression, in both normal and heat‐shock stress environments.
Single‐step purification of a protein‐folding catalyst, the SlyD peptidyl prolyl isomerase (PPI), from cytoplasmic extracts of Escherichia coli
Authors: Mukherjee Sourav , Shukla Anshuman , Guptasarma Purnananda
Published: October 2003

The protein‐folding catalyst SlyD, a peptidyl prolyl cis –trans isomerase regulated by metal binding, was initially discovered as a major contaminant of non‐denaturing immobilized metal‐affinity chromatography (IMAC)‐based procedures for the purification of heterologously expressed 6×His‐tagged proteins from Escherichia coli . Given its ability to bind weakly to nickel‐nitrilotriacetic acid (Ni2+‐NTA), protocols for the purification of SlyD currently use an initial non‐denaturing IMAC step, followed by an ion‐exchange chromatographic step and occasionally also other chromatographic steps. Here we demonstrate that using denaturing conditions instead of non‐denaturing conditions, and by processing of large quantities of culture through small volumes of Ni2+‐NTA resin to increase competition for binding, single‐step purification of SlyD to homogeneity can be achieved directly from E. coli extracts, as assessed through spectroscopic and electrophoretic methods. The purified, denatured SlyD is shown to be capable of refolding to give rise to a native‐like CD spectrum, establishing the utility of the procedure. The procedure also establishes SlyD to be the only E. coli protein capable of contaminating denaturing IMAC‐based procedures.
Enzymic cleavage of fusion protein using immobilized urokinase covalently conjugated to glyoxyl‐agarose
Authors: Suh Chang Woo , Choi Gang Sun , Lee Eun Kyu
Published: October 2003

We immobilized urokinase (UK) by covalent attachment to activated Sepharose 6B‐CL through multi‐point amine coupling and evaluated its performance in cleaving a fusion protein, which consisted of recombinant human growth hormone (hGH) and a fragment of glutathione S‐transferase that was linked by a tetrapeptide of a UK‐specific recognition sequence. Packing densities of aldehyde groups on the activated agarose surface could be controlled in a gel range of 7–60 μ mol/ml aldehyde by the amount of glycidol used. The immobilization yield was nearly 100% at pH 10.5, and the specific activity of the immobilized UK was equivalent to about 80% of soluble UK under the assay conditions. The immobilized UK showed an improvement in pH and thermal stability, probably due to the structural rigidity imparted by multi‐point linkages to the matrix. The cleavage rate by the immobilized UK was lower than that of the soluble enzyme but the side reaction of cryptic cleavage was significantly decreased, which might suggest that the enzyme's specificity was altered by the immobilization. Cleavage yield in the column packed with immobilized UK was dependent on the feed rate, and the yield was approx. 80% of that of the soluble UK. The monomeric hGH could be obtained by selectively precipitating the uncleaved fusion protein and the GST fragments at an acidic pH.
Enhanced production of verbenol, a highly valued food flavourant, by an intergeneric fusant strain of Aspergillus niger and Penicillium digitatum
Authors: Rao Smitha C. V. , Rao Rati , Agrawal Renu
Published: October 2003

An intergeneric hybrid, obtained from Aspergillus niger and Penicillium digitatum, biotransformed (−)‐cis ‐α ‐pinene (5 mg/25 ml) into (−)‐cis ‐verbenol (60%; 1.08 mg/g of biomass) in 6 h compared with 0.18 mg/g of biomass for verbenol (10–15%; 0.18–0.27 mg/g of biomass) in the initial parent cultures.
Overexpression of the pectin lyase gene of Pseudomonas marginalis in Escherichia coli and purification of the active enzyme
Authors: Papi Rigini , Kyriakidis Dimitrios
Published: October 2003

A pectin lyase gene (pnl) of Pseudomonas marginalis was cloned and overexpressed in Escherichia coli BL21(DE3). The pnl gene was amplified by PCR, inserted into pET29c with a six‐His tag and the overproduced active enzyme was purified almost to homogeneity using a Ni2+‐nitrilotriacetate–agarose column. The purified pectin lyase (PNL; EC 4.2.2.10, family 1) is inhibited by NAD+ (at concentrations above 0.25 mM), NADH or dithiothreitol. Evidence for the existence of a heat‐labile protein inhibitor of PNL is also reported. The DNA‐binding ability of PNL was demonstrated by DNA‐retardation experiments. The partially purified enzyme was incubated with plasmid DNA and the complex was shifted to a higher molecular mass. Analysis of the electroeluted proteins from the protein–DNA complex revealed that one of the electroeluted protein bands was PNL. Antibodies against the overexpressed PNL were also prepared and partially purified.
Kinetic study of recombinant human protein disulphide isomerase‐assisted C125A recombinant human interleukin‐2 folding
Authors: Du Chengan , Wolfe Janet L.
Published: October 2003

A kinetic model was developed to describe recombinant human protein disulphide isomerase (rhPDI)‐assisted folding of a substrate protein, C125A recombinant human interleukin‐2 (C125A rhIL‐2). A series of progress curves showing native C125A rhIL‐2 formation under different reaction conditions were generated. Non‐linear regression analysis of the progress curves of rhPDI‐assisted C125A rhIL‐2 folding was used to fit the differential equations of the described kinetic models. The goodness‐of‐fit of the model to the experimental datasets was used to support or exclude a particular kinetic model of rhPDI‐assisted C125A rhIL‐2 folding. The results suggest that the formation of native C125A rhIL‐2 results from both glutathione‐dependent oxidative folding and rhPDI‐catalysed folding reactions. During oxidative folding of C125A rhIL‐2, both rhPDI and reduced C125A rhIL‐2 aggregated in folding buffer. The aggregation rates of rhPDI and C125A rhIL‐2 followed second‐order kinetics. Guanidinium chloride inactivated rhPDI but also decreased the aggregation of reduced C125A rhIL‐2. These results demonstrate that during rhPDI‐assisted C125A rhIL‐2 folding, reduced C125A rhIL‐2 aggregation competes with the productive folding pathway. While rhPDI enhances the oxidative folding of C125A rhIL‐2, inactivation of rhPDI by the residual guanidinium chloride compromises its catalytic efficiency. The established model can be used to optimize the folding components in the folding mixture, and thus improve the folding efficiency.
Lipase assays for conventional and molecular screening: an overview
Authors: Gupta Rani , Rathi Pooja , Gupta Namita , Bradoo Sapna
Published: October 2003

Lipases are versatile biocatalysts that can perform innumerable different reactions. Their enantio‐, chemo‐ and stereo‐selective nature makes them an important tool in the area of organic synthesis. Unlike other hydrolases that work in aqueous phase, lipases are unique as they act at the oil/water interface. Besides being lipolytic, lipases also possess esterolytic activity and thus have a wide substrate range. Hence, the lipase assay protocols hold a significant position in the field of lipase research. Lipase activity can be estimated using a wide range of assay protocols that differ in terms of their basic principle, substrate selectivity, sensitivity and applicability. As the value of these enzymes continues to grow and new markets are exploited, development of new or improved enzymes will be a key element in the emerging realm of biotechnology. Hence, development of faster and simpler protocols incorporating newer and more specific substrates is the need of the hour. In this endeavour, methods that could be adopted for molecular screening occupy an important position. Here, an overview of the lipase assay protocols is presented with emphasis on the assays that can be adopted for the molecular screening of these biocatalysts.
Cissus sicyoides (princess vine) in the long‐term treatment of streptozotocin‐diabetic rats
Authors: Pepato Maria Teresa , Baviera Amanda Martins , Vendramini Regina Célia , Da Silva Perez Maira da Penha Marques , Kettelhut Isis do Carmo , Brunetti Iguatemy Lourenço
Published: October 2003

Leaf decoctions of Cissus sicyoides (princess vine) are taken widely as a popular remedy for diabetes mellitus in Brazil, where its common name is ‘vegetal insulin’. However, there have been practically no attempts so far to determine scientifically whether it has anti‐diabetic effects and we decided to administer leaf decoctions, over extended periods, to normal and streptozotocin‐diabetic rats, and investigate the effects of this treatment on the physiological and metabolic parameters that are altered in diabetic animals. The experimental model adopted was shown to be appropriate by running a parallel treatment with insulin, which led to expected improvements in several abnormal parameter values. The decoction treatment significantly reduced the intake of both food and fluid and the volume of urine excreted, as well as the levels of blood glucose, urinary glucose and urinary urea, in comparison with controls. Lipid metabolism was not affected by the treatment; nor was the level of hepatic glycogen in diabetic animals, which indicated that the mechanism responsible for the improvement in carbohydrate metabolism, observed in animals treated with the decoction, could not involve inhibition of glycogenolysis and/or stimulation of glycogenesis. The fact that normal animals treated with C. sicyoides exhibited no changes in any of the measured parameters suggests that its mode of action in diabetic animals does not resemble those of sulphonylurea or insulin. It may, however, act in a similar way to biguanide, via inhibition of gluconeogenesis.
Process characterization of the chromatographic steps in the purification process of a recombinant Escherichia coli‐expressed protein
Authors: Rathore Anurag S. , Johnson Gary V. , Buckley John J. , Boyle Denis M. , Gustafson Mark E.
Published: October 2003

Process development and characterization studies were performed for the chromatographic steps in the purification process of a recombinant Escherichia coli‐expressed protein product candidate. The objective of this work was to develop a robust and efficient purification process that would generate material of adequate purity and quantity. A resin screening procedure was developed to aid in picking out the optimal resin for each of the chromatographic columns. It was found that, as a result of resin screening, it was possible to come up with a process with only two column‐chromatographic steps. The resulting process used a sulphopropyl (SP) and a quaternary amino (Q) column with intermittent ultrafiltration steps for purification. Effects of different process parameters such as the gradient slope, pH, flow velocity and protein loading on the column performance were evaluated. Buffer pH for the SP column, and buffer pH, gradient slope, protein loading and flow velocity for the Q column, were identified as parameters that could have a significant impact on the performance of the chromatographic step and would require further characterization to improve the robustness of the process. Further process characterization led to the findings that the gradient slope, load pH and buffer pH of the Q column have a significant impact on column performance (>15% change in step yield). All other parameters under consideration did not have any significant impact on pool quality (>10% change in pool purity for the SP column and >5% for the Q column). On the basis of small‐scale studies, optimum operating conditions were chosen and the purification process was successfully scaled‐up to a large‐scale robust process with step yields and product quality that were better than those at the small scale.