bluuebunny/crossref_metadata_2025 huggingface dataset:
α2‐Adrenergic receptor binding in canine Purkinje fibers
Authors: Lee Hon-Chi , Samson Ricardo A. , Cai John J.
Published: April 2003

Autoradiograms were developed from free running strands of Purkinje fibers and from left ventricular myocardium of dog hearts exposed to [7‐methoxy‐3H]prazosin or [O‐methyl‐3H]yohimbine in the presence or absence of excessive non‐radioactive ligands. Both Purkinje fiber and ventricular myocardium showed high density, tissue‐specific binding to [3H]prazosin. In contrast, high density, tissue‐specific binding to [3H]yohimbine was present in Purkinje fibers but not in ventricular myocardium. Membrane fractions showed high affinity, saturable, and displaceable binding with [3H]yohimbine in preparations from canine cardiac Purkinje fibers but not those from canine cardiac ventricular myocardium. Scatchard analysis of the canine Purkinje membrane α
2‐adrenergic receptor binding showed a B
max of 54.9 fmol/mg protein with a K
d of 6.25 nM. These results confirm the electrophysiological findings that post‐junctional α
2‐adrenergic receptors are present in Purkinje fibers.
Sensitive substrates for neprilysin (neutral endopeptidase) and thermolysin that are highly resistant to serine proteases
Authors: Spungin-Bialik Anya , Ben-Meir Daniella , Fudim Ella , Carmeli Shmuel , Blumberg Shmaryahu
Published: April 2003

Tripeptide derivatives like 3‐carboxypropanoylalanyl‐alanyl‐leucine 4‐nitroanilide or 3‐carboxypropanoylalanyl‐alanyl‐phenylalanine 4‐nitroanilide are very sensitive substrates for neprilysin (k
cat > 102 s−1; k
cat/K
m ≥ 106 s−1 · M−1) and are widely employed in investigations of the enzyme. However, these compounds are also good substrates for the serine proteases chymotrypsin and subtilisin (k
cat ∼ 1s−34 s−1). By substituting the N‐terminal alanine of the substrates with proline, the catalytic efficiency of the enzymic reaction, by the serine proteases, is diminished by 2–3 orders of magnitude, whereas that by neprilysin and thermolysin decreases only slightly. These effects demonstrate that structural alterations in peptide substrates that impair secondary sub‐site interactions with one class of peptidases may enhance the selectivity of the substrates towards another class of peptidases.
A role for ADP‐ribosylation factor 1, but not COP I, in secretory vesicle biogenesis from the trans‐Golgi network
Authors: Barr Francis A. , Huttner Wieland B.
Published: April 2003

A synthetic N‐myristoylated peptide corresponding to the amino‐terminal domain of ADP‐ribosylation factor 1 (ARF1) markedly increases, in a cell‐free system using post‐nuclear supernatant from PC12 cells, the biogenesis of constitutive secretory vesicles and immature secretory granules from the trans‐Golgi network (TGN). The related N‐myristoylated ARF4 peptide only weakly stimulates, and the non‐myristoylated ARF1 and ARF4 peptides inhibit, the biogenesis of these secretory vesicles. In a modified cell‐free system using TGN membranes, coatomer‐depleted cytosol supports the biogenesis of TGN‐derived secretory vesicles to the same extent as control cytosol. These results suggest a role for ARF1, but not the COP I coat, in secretory vesicle biogenesis from the TGN, possibly via the activation of phospholipase D.
Human mineralocorticoid receptor interacts with actin under mineralocorticoid ligand modulation
Authors: Jalaguier Stéphan , Mornet Dominique , Mesnier Danielle , Léger Jean J. , Auzou Gilles
Published: April 2003

The human mineralocorticoid receptor of the steroid receptor family contains a modular structure with domain E which is considered to be a hormone binding domain. Recombinant protein approaches enabled us to clearly determine that this domain is also able to interact with F‐actin (K
d about 2 μM) and G‐actin. Moreover, it was revealed that this mineralocorticoid receptor domain/actin interaction was modulated by specific mineralocorticoid ligands. Agonist (aldosterone) steroid binding almost totally (91%) abolished the interaction with F‐actin, while antagonist (progesterone) binding allowed more than 30% of this binding. Steroid modulation of the interaction between domain E and actin indicated that this actin binding is specific and could be essential for cellular mineralocorticoid receptor activity.
Site‐directed mutagenesis of the base recognition loop of ribonuclease from Bacillus intermedius (binase)
Authors: Okorokov Andrei L. , Panov Konstantin I. , Kolbanovskaya Elena Yu. , Karpeisky Marat Ya. , Polyakov Konstantin M. , Wilkinson Anthony J. , Dodson Guy G.
Published: April 2003

Members of the microbial guanyl‐specific ribonuclease family show a high level of structural homology. The structural basis for guanyl base binding by microbial ribonucleases has been established for all members of the family and the existence of a guanine recognition loop was shown. However, bacillar RNases such as binase and barnase show far less specificity towards the guanyl base in hydrolysing oligonucleotides composed of more than 4 or 5 nucleotides. Using site‐directed mutagenesis we introduced a number of amino acid substitutions into the base recognition loop of binase. The donor sequence originated from the guanyl specific ribonuclease Sa. Two single, two double and one triple (entire loop substitution) mutants were constructed and overproduced in E. coli. The kinetic properties of the mutant variants are different from the wild‐type protein. Amino acid substitutions R61V, G60S, S56Q/R61V, G60S/R61V show 3‐fold, 7‐fold, 4‐fold and 12‐fold increased guanyl specificity respectively. However, all mutants retain the ability to catalyse the hydrolysis of a poly(A) substrate.
Calcineurin and mitochondrial function in glutamate‐induced neuronal cell death
Authors: Ankarcrona Maria , Dypbukt Jeannette M. , Orrenius Sten , Nicotera Pierluigi
Published: April 2003

We have previously reported that glutamate can trigger a succession of necrosis and apoptosis in cerebellar granule cells (CGC). Since specific blockers of the (NMDA) receptor channel prevented both types of cell death, the role of Ca2+‐dependent processes in the initiation of glutamate toxicity was further investigated. We examined the possible involvement of mitochondria and the role of the Ca2+/calmodulin‐regulated protein phosphatase, calcineurin, in the development of either type of cell death. Cyclosporin A and the more selective calcineurin inhibitor, FK‐506, prevented the development of both early necrosis and delayed apoptosis. In addition, cyclosporin A prevented the collapse of mitochondrial membrane potential observed during the exposure to glutamate and the concomitant necrotic phase. When CsA was added immediately after glutamate removal, it also prevented delayed apoptosis of neurons that had survived the necrotic phase. Altogether, these results suggest the involvement of calcineurin and a role for mitochondrial deenergization as early signals in neuronal apoptosis induced by glutamate.
Reciprocal control of the conformational state of the sarcoplasmic reticulum calcium channel protein by polarization and depolarization in the transverse tubule
Authors: Ikemoto Noriaki , El-Hayek Roque
Published: April 2003

We attached the conformational probe methylcoumarin acetate (MCA) specifically to the junctional foot protein (JFP) moiety of triads, and monitored conformational changes in the JFP during polarization and depolarization of the T‐tubule moiety. The MCA fluorescence decreased upon T‐tubule polarization, and the fluorescence changes were blocked by preventing T‐tubule polarization or by a nimodipine block of the T‐tubule‐to‐sarcoplasmic reticulum communication. Depolarization of the T‐tubule reversed the MCA fluorescence decrease which had been produced by T‐tubule polarization. These results suggest that the conformational and functional states of the JFP are regulated by T‐tubule polarization and depolarization in a reciprocal fashion.
Molecular cloning of frog secretogranin II reveals the occurrence of several highly conserved potential regulatory peptides
Authors: Anouar Youssef , Jégou Sylvie , Alexandre David , Lihrmann Isabelle , Michael Conlon J. , Vaudry Hubert
Published: April 2003

Secretogranin II (SgII) is an acidic secretory protein present in large dense core vesicles of neuronal and endocrine cells. Based on the sequence of a peptide derived from the processing of SgII in the brain of the frog Rana ridibunda, degenerate oligonucleotides were used to clone the cDNA encoding frog SgII from a pituitary cDNA library. This cDNA encodes a 574 amino acid protein which exhibits 46–48% sequence identity with mammalian SgII and contains 11 pairs of basic amino acids. Four potential processing products delimited by pairs of basic residues exhibited a much higher degree of identity (68–82%) with the corresponding mammalian SgII sequences. The frog SgII mRNA is ∼4 kb in length and is differentially expressed in the brain and endocrine tissues. The present data reveal that several SgII‐derived peptides have been highly conserved during evolution, suggesting that these peptides may play important neuroendocrine regulatory functions.
Modulatory effect of extracellular Mg2+ ions on K+ and Ca2+ currents of capillary endothelial cells from rat brain
Authors: Delpiano Marco A. , Altura Burton M.
Published: April 2003

Using whole‐cell patch‐clamp recording, we demonstrate that exposure of single rat brain capillary endothelial cells to different extracellular Mg2+ concentrations (0.3, 4.8 and 9.6 mM) affects the conductance of K+ and Ca2+ currents elicited under control conditions (1.2 mM). Extracellular Mg2+ concentrations ([Mg2+]o) of 4.8 and 9.6 mM reversibly depress outward K+ currents by about 30 ± 12% (n =10) and 3% ± 13%(n = 10), at all activating potentials, respectively. Using identical concentrations reversibly depressed the Ca2+ current by about 40 ± 16% (n = 8) and 46 ± 18% (n = 6), respectively. Using a low Mg2+ concentration of 0.3 mM, the K+ current activation was unexpectedly and mildly increased by about 15 ± 5% (n = 5), and the inward Ca2+ current was attenuated. When studying this effect of low [Mg2+]o on ‘pure’ Ca2+ currents, free of outward currents, we found that this inward current was depressed by about 38 ± 16% (n = 8), and its threshold for activation, in the current‐voltage relationship, was shifted to more negative potentials. It is concluded that high [Mg2+]o hinders the entry of Ca2+ through low‐voltage activated Ca2+ channels and thereby attenuates a Ca2+‐regulated K+ conductance. At a low [Mg2+]o (0.3 mM), Mg2+ shifts the steady‐state inactivation of the voltage‐activated Ca2+ channel to more negative potentials by about 8 mV (n = 6), probably due to a negative screening effect, i.e. a reduction of positive charges on the cell membrane. This may contribute to an apparent increase in K+ conductance by an, as yet, unknown mechanism.
Histidine 131, not histidine 43, of the Bradyrhizobium japonicum FixN protein is exposed towards the periplasm and essential for the function of the cbb3‐type cytochrome oxidase
Authors: Zufferey Rachel , Thöny-Meyer Linda , Hennecke Hauke
Published: April 2003

In subunit I (FixN) of the Bradyrhizobium japonicum cbb
3‐type oxidase, only five instead of the normal six strictly conserved histidines (H) could be unambiguously assigned as the putative heme or copper ligands. The ambiguity concerned H43 or H131 as the presumptive N‐terminal ligands of the low‐spin heme B. We report here that a H43A replacement had a wild‐type phenotype, whereas the H131A mutant was defective in oxidase function and subunit assembly or stability, suggesting that H131 serves as the N‐terminal low‐spin heme ligand. Topological studies revealed that H131 resides on the periplasmic side of helix 2, where one of the low‐spin heme ligands is normally found in conventional heme‐copper oxidases.
Effects of synaptic vesicles on actin polymerization
Authors: Chieregatt E. , Ceccaldi P.E. , Benfenati F. , Valtorta F.
Published: April 2003

We have analyzed the effects of synaptic vesicles on actin polymerization by using a time‐resolved spectrofluorometric assay. We have found that synaptic vesicles have complex effects on the kinetics of actin polymerization, which vary depending on whether the synaptic vesicle‐specific phosphoprotein synapsin I is absent or present on their membrane. Synapsin I bound either to synaptic vesicles or to pure phospholipid vesicles exhibits phosphorylation‐dependent actin‐nucleating activity. Synaptic vesicles depleted of endogenous synapsin I decrease the rate and the final extent of actin polymerization, an effect which is not observed with pure phospholipid vesicles. Thus, the state of association of synapsin I with synaptic vesicles, which is modulated by its state of phosphorylation, may affect actin assembly and the physico‐chemical characteristics of the synaptic vesicle microenvironment.
Is the Glass Half Full or Half Empty?: Reflections on Five Years of Competitive Politics in Eastern Europe
Authors: Comisso Ellen
Published: April 2003

This article explores the causes for the pervasive sentiment in contemporary Eastern Europe to the effect that “it is democratic but it isn't”. It begins with a discussion of the differences between “procedural” and “substantive” democracy, and finds that the institutions of procedural democracy have proven surprisingly robust in the post-communist period. It then examines three variants of substantive democracy: liberal, national, and egalitarian. Each links democratic government to a particular social and economic order, none of which fully describes the actual situation within most states in the area. Finally, these distinctions between types of democracy are employed to illuminate the dynamics of both policy choices regarding restitution and privatization and electoral outcomes in the past half-decade.
Expanding Political Space for Women in Poland: An Analysis of Three Communities
Authors: Graham Ann , Regulska Joanna
Published: April 2003

The paper argues that Poland's emerging democracy has yet to open political space for women. Through an analysis of three case study communities, the authors that Poland's political culture—whether communist or democratic—cannot be relied on to ensure women's equal participation. Formal mechanisms of power continue to favor the experience of men. By focusing on the ignored context of women's local activism, the study draws attention to the barriers and opportunities for women and how these barriers play out differently in which political cultures are similar yet distinct.
The End of Liberalism?: Economic Liberalization and the Transformation of Post-Communist Poland
Authors: Zubek Voytek
Published: April 2003

Until the mid-1980s the overwhelming majority of the opposition's leaders simply believed that “socialism was just a very good thing.” The only problem was that the opposition viewed the socialism that was constructed by the Communist party as distorted by systemic and ideological errors. From the early 1980s the first seeds of economic liberalism were attempting to sprout on what seemed to be a definitely hostile and infertile field since the Leftist weltunschauung dominated the minds and “...souls of the opposition's elite.” As a result, economic liberalism was rediscovered by some elements belonging to an entirely new generation of anti-Communist opposition. It first became popular within narrow circles of young economists centered in Warsaw, Gdansk and Cracow. Perhaps these groups would have remained on the social and intellectual margin if any of the socialistic schemes to rectify the systemic problems of the Polish “real socialism” had succeeded. With the failure of these various schemes, Poland's reformed Communists began to entertain other systemic options. While some of them still continued their attempts to carve out the uniquely Polish “Third Way” of combining socialism with petty-entrepreneurialism and democracy, others began to look more seriously at some systemic solutions that were influenced by economic liberalism. Eventually, in the second half of the 198Os, the Communist elite began to cross the ideological Rubikon that divided the statist/socialist from the free market/entrepreneurial weltanschauung. Against this sociopolitical background, this paper aims to explore the gradual emergence of economic liberalism in Poland in the 1980s and the elusive role that it played in the society's systemic transformation.
Votes, seats and power: 1996 Parliamentary election in the Czech Republic
Authors: Turnovec František
Published: April 2003

This paper gives a brief survey of the results of the 1996 election to the Lower House and the Upper House of the Parliament of the Czech Republic. Methodology of power indices is used to evaluate voting power and relative influence of the parties represented in the parliament. A comparison with the 1992 electoral results is provided. The regional dimension of the electoral results is discussed, and a hypothetical impact of different values of electoral threshold on the structure of parliament is analyzed.
A comparative analysis of leveraged recapitalization versus leveraged buyout as a takeover defense
Authors: Bae Sung C. , Simet Daniel P.
Published: April 2003

Two types of defensive scheme—leveraged buyout (LBO) and leveraged recapitalization (LR)—are examined. In particular, this article examines (1) whether the two similar defensive tactics affect stockholder returns differently and (2) what firm attributes are associated with stockholder gains in LBO and LR announcements. This study finds that stocks of both LBO and LR firms, on average, exhibit significant positive abnormal returns during the announcement period, but that the latter experience substantially smaller returns than the former. This study further finds that while mitigation of agency problems associated with a firm's free cash flow is present for both LR and LBO firms, it is more pronounced for the LBO firms. These results provide evidence that a firm with higher free cash flow could benefit a greater reduction of agency costs by going private through a LBO plan than by remaining public through a LR plan.
Can insiders bail themselves out before private renegotiation?
Authors: Yur‐Austin Jasmine
Published: April 2003

This study examines insider trading for a sample of firms that announce a workout agreement, controlling for both successful and unsuccessful workout attempts. I find that insider trading activity is related to the outcome of the workout proposal. Managers tend to bail out (sell shares) of firms that are unsuccessful in the workout process while they purchase shares prior to the workout announcements if the firms are ultimately successful in their workout. In addition, the evidence suggests managerial trading behavior is related to the workout market reaction. When a workout announcement is preceded by insider buying (selling), the stock price reaction is positive (negative). Overall, the evidence presented in this article is consistent with the notion that insider transactions convey private information to the stock market about a financially distressed firm.
Severe Gestational Edema
Authors: Reynolds Dianne
Published: April 2003

Lower extremity edema is an almost universal finding in late pregnancy, occurring secondary to increased venous pressure in the legs, obstruction of lymphatic flow, and reduced plasma colloid osmotic pressure.1 Varney describes gestational edema as the development of an excessive accumulation of fluid in the tissues without coexisting hypertension or proteinuria.2 Midwives are familiar with this common condition in an otherwise uncomplicated pregnancy and are comfortable reassuring clients that lower extremity edema is non‐pathologic. The patient with severe lower extremity edema, however, presents the provider with a clinical challenge. Is the presenting symptom a sign of an underlying disease process, or simply a normal physiologic process with extreme manifestation/expression? Lower extremity edema that rises to include the labia presents an intrapartum challenge of managing tissue integrity during the birth. A case of severe gestational edema is reported. Discussion explores broader considerations in differential diagnosis and management.
New Directions in Midwifery Education: The Master's of Science in Midwifery Degree
Authors: Farley Cindy , Carr Kathy Camacho
Published: April 2003

Midwifery is reclaiming its perspective as a discipline separate from, yet integrally related to nursing and medicine. Emerging trends in health care place increased demands on the knowledge base and clinical practice of midwifery, stimulating a need for new directions in midwifery education. The master's of science with a major in midwifery is a new degree option available to midwifery students in the United States. This article presents the argument that midwifery is a distinct discipline and describes the importance of a master's of science in midwifery degree toward furthering the work of the discipline of midwifery. Descriptions of the current master's of science in midwifery degree programs are included.
Die affektive Bindung an das Unternehmen
Authors: Maier Günter W. , Woschée Ralph-Michael
Published: April 2003

Zusammenfassung. In dieser Studie wurden die psychometrischen Eigenschaften und die Validität einer deutschsprachigen Fassung des Organizational Commitment Questionnaire (OCQ) überprüft. Die Studie wurde durchgeführt mit 1021 ehemaligen Hochschulabsolventen, die zum Zeitpunkt der Befragung seit durchschnittlich 1,9 Jahren berufstätig waren. Außer dem OCQ wurden weitere Arbeitseinstellungen erhoben (Arbeitszufriedenheit und Job Involvement), Einschätzungen zu mehreren Arbeitsplatzmerkmalen (z. B. Handlungsspielraum), zur Organisation (Ausmaß der Zielübereinstimmung) und zur individuellen Gewichtung der Arbeitstätigkeit (Karriere- vs. Freizeitorientierung) sowie zur Kündigungsabsicht. Eine konfirmatorische Faktorenanalyse bestätigte die einfaktorielle Struktur des OCQ im Vergleich zu zweifaktoriellen Modellen. Die diskriminante Validität des OCQ konnte dadurch demonstriert werden, dass er sich von den anderen Arbeitseinstellungen faktorenanalytisch gut unterscheiden ließ und die Arbeitseinstellungen jeweils unterschiedliche spezifische Zusammenhänge mit den weiteren Variablen aufwiesen.
A Comparison of the Contrast Effects in Sound Localization in the Horizontal and Vertical Planes
Authors: Getzmann Stephan
Published: April 2003

Abstract. The effect of a background sound on the auditory localization of a single sound source was examined. Nine loudspeakers were arranged crosswise in the horizontal and the median vertical plane. They ranged from -20° to +20°, with the center loudspeaker at 0° azimuth and elevation. Using vertical and horizontal centimeter scales, listeners verbally estimated the position of a 500-ms broadband noise stimulus being presented at the same time as a 2 s background sound, emitted by one of the four outer loudspeakers. When the background sound consisted of continuous broadband noise, listeners consistently shifted the apparent target positions away from the background sound locations. This auditory contrast effect, which is consistent with earlier findings, equally occurred in both planes. But when the background sound was changed to a pulse train of noise bursts, the contrast effect decreased in the horizontal plane and increased in the vertical plane. This discrepancy might be due to general differences in the processing of interaural and spectral localization information.
Individual Unionization Decisions: A Multilevel Model of Cost-Benefit Influences
Authors: Mellor Steven , Holzworth R. James , Conway James M.
Published: April 2003

Abstract. An important part of the unionization process is predicting the individual’s vote for or against union representation. We proposed and tested a multilevel model based on the relative importance of costs and benefits of representation. Regression statistics from within-person analyses were used to show the influence of perceived costs and benefits on judgment policies about intent to vote in a representation election. Using these statistics as outcome variables, between-person analyses were used to show the influence of decision frame on cost-benefit influences, in which framing a vote as one for or against the union was conceived as a contextual variable in an election. Results were used to extend prior unionization research and to suggest how employers and unions may attend to framing effects in an election.
Adenosine 5′‐triphosphate (ATP) is an excitatory cotransmitter with noradrenaline to the smooth muscle of the rat prostate gland
Authors: Ventura Sabatino , Dewalagama Ramila K , Lau Linda C L
Published: April 2003

This study investigated whether adenosine 5′‐triphosphate (ATP) is involved in neurotransmission to the rat prostate gland.

Fluorescence immunohistochemistry carried out on formaldehyde‐fixed and frozen sections of rat prostate showed immunoreactivity for the P2X1‐receptor in the fibromuscular stroma surrounding the secretory acini but not in the glandular epithelium. P2X2‐, P2X3‐, P2X4‐ and P2X7‐receptors were immunonegative in the rat prostate stroma. Double‐staining procedures showed P2X1‐receptor immunoreactivity to be colocalized with α‐actin immunoreactivity.

Isolated organ bath studies investigated whether drugs, which modify purinergic mechanisms, are able to affect contractility of the rat prostate gland. Suramin (100 μM) and αβmethylene ATP (10 μM) inhibited contractile responses to trains of electrical‐field stimulation (70 V, 0.5 ms, 0.1–2 Hz) in the absence and presence of prazosin (300 nM). Responses to 5–20 Hz were unaffected by suramin or αβmethylene ATP.

Exogenous application of ATP analogues to unstimulated isolated preparations of rat prostate produced concentration‐dependent suramin (100 μM) sensitive transient contractions with a relative order of potency: αβmethylene ATP>βγmethylene ATP>ATP.

Adenosine and adenosine 5′‐monophosphate (AMP) did not produce contractile responses.

These results suggest that P2X1‐receptors for ATP, which mediate contractions are present in the fibromuscular stroma of the rat prostate. The relative order of potency of ATP analogues in producing contractions of the rat prostate is consistent with the activation of P2X1‐receptors. Inhibition by suramin and αβmethylene ATP of electrically evoked nerve‐mediated contractions of the rat prostate implies that ATP contributes to this contractile response and is therefore a cotransmitter with noradrenaline during low‐frequency stimulation.

British Journal of Pharmacology (2003) 138, 1277–1284. doi:10.1038/sj.bjp.0705167
Opposite nociceptive effects of the arginine/NO/cGMP pathway stimulation in dermal and subcutaneous tissues
Authors: Vivancos Gustavo G , Parada Carlos A , Ferreira Sérgio H
Published: April 2003

Nitric oxide has been described either as pronociceptive or antinociceptive. In this investigation, using an electronic pressure‐metre, the intradermal and the subcutaneous effects of prostaglandin E2 (PGE2) and agents that mimic or inhibit the arginine/NO/cGMP pathway were compared.

The hypernociceptive effect of the intradermal injection of PGE2 (100 ng) was immediate, peaking within 15–30 min and returning to basal values in 45–60 min. The subcutaneous injection of PGE2 induced a hypernociception with a delayed peak (3 h) plateauing for 4–6 h.

Intradermal administration of 3‐morpholino‐sydnonimine‐hydrochloride (SIN‐1) enhanced, while its subcutaneous administration inhibited, subcutaneous hypernociception induced by PGE2. This inhibition was prevented by ODQ (8 μg) but not by NG‐monomethyl‐L‐arginine (L‐NMMA) (50 μg).

Intradermal but not subcutaneous administration of L‐arginine (1–100 μg), SIN‐1 (1–100 μg) and dibutyrylguanosine 3′:5′‐cyclic monophosphate (db cGMP) (0.1–100 μg) induced an early (15–30 min) dose‐dependent hypernociceptive effect. Intradermal pretreatment with NG‐monomethyl‐L‐arginine (L‐NMMA; 50 μg) inhibited the hypernociception induced by L‐Arg (10 μg), but not that induced by SIN‐1 (10 μg) or db cGMP (10 μg).

Intradermal injection of ODQ (8 μg) antagonized the hypernociception induced by L‐arginine and SIN‐1, but not that induced by db cGMP.

Considering (a) the different time course of intradermal and subcutaneous PGE2‐induced hypernociception, (b) the opposite nociceptive effect of intradermal and subcutaneous administration of SIN‐1 (db cGMP) as well as the arginine/NO/cGMP pathway, the existence of different subsets of nociceptive primary sensory neurons in which the arginine/NO/cGMP pathway plays opposing roles is suggested. This hypothesis would explain the apparent contradictory observations described in the literature.

British Journal of Pharmacology (2003) 138, 1351–1357. doi:10.1038/sj.bjp.0705181
A stateful multicast access control mechanism for future metro‐area‐networks
Authors: Sun Wei‐qiang , Li Jin‐sheng , Hong Pei‐lin
Published: April 2003

Multicasting is a necessity for a broadband metro‐area‐network. Discusses the security problems with current multicast protocols. To make the multicast delivery infrastructure more secure and reliable, a stateful multicast access control mechanism, based on MAPE, is proposed. This mechanism expands the current forwarding procedure of a layer 2 switch so that stateful multicast access control can be carried out at the very edge of the metro area network. The architecture of MAPE is discussed in detail, as well as the states maintained and messages exchanged. Further explains that the scheme is flexible and scalable.
Functional Activity of Antibodies against the Recombinant OpaJ Protein fromNeisseria meningitidis
Authors: de Jonge M. I. , Vidarsson G. , van Dijken H. H. , Hoogerhout P. , van Alphen L. , Dankert J. , van der Ley P.
Published: April 2003

The opacity proteins belong to the major outer membrane proteins of the pathogenicNeisseriaand are involved in adhesion and invasion. We studied the functional activity of antibodies raised against the OpaJ protein from strain H44/76. Recombinant OpaJ protein was obtained fromEscherichia coliin two different ways: cytoplasmic expression in the form of inclusion bodies followed by purification and refolding and cell surface expression followed by isolation of outer membrane complexes (OMCs). Immunization with purified protein and Quillaja saponin A (QuilA) induced high levels of Opa-specific antibodies, whereas theE. coliOMC preparations generally induced lower levels of antibodies. Two chimeric Opa proteins, hybrids between OpaB and OpaJ, were generated to demonstrate that the hypervariable region 2 is immunodominant. Denatured OpaJ with QuilA induced high levels of immunoglobulin G2a (IgG2a) in addition to IgG1, whereas refolded OpaJ with QuilA induced IgG1 exclusively. These sera did not induce significant complement-mediated killing. However, all sera blocked the interaction of OpaJ-expressing bacteria to CEACAM1-transfected cells. In addition, cross-reactive blocking of OpaB-expressing bacteria to both CEACAM1- and CEA-transfected cells was found for all sera. Sera raised against purified OpaJ and against OpaJ-containing meningococcal OMCs also blocked the nonopsonic interaction of Opa-expressing meningococci with human polymorphonuclear leukocytes.
Invasion and Killing of Human Endothelial Cells by Viridans Group Streptococci
Authors: Stinson Murray W. , Alder Susan , Kumar Sarmishtha
Published: April 2003

Colonization of the cardiovascular endothelium by viridans group streptococci can result in infective endocarditis and possibly atherosclerosis; however, the mechanisms of pathogenesis are poorly understood. We investigated the ability of selected oral streptococci to infect monolayers of human umbilical vein endothelial cells (HUVEC) in 50% human plasma and to produce cytotoxicity. Planktonic
Streptococcus gordonii
CH1 killed HUVEC over a 5-h period by peroxidogenesis (alpha-hemolysin) and by acidogenesis but not by production of protein exotoxins. HUVEC were protected fully by addition of supplemental buffers and bovine liver catalase to the culture medium. Streptococci were also found to invade HUVEC by an endocytic mechanism that was dependent on polymerization of actin microfilaments and on a functional cytoskeleton, as indicated by inhibition with cytochalasin D and nocodazole. Electron microscopy revealed streptococci attached to HUVEC surfaces via numerous fibrillar structures and bacteria in membrane-encased cytoplasmic vacuoles. Following invasion by
S. gordonii
CH1, HUVEC monolayers showed 63% cell lysis over 4 h, releasing 64% of the total intracellular bacteria into the culture medium; however, the bacteria did not multiply during this time. The ability to invade HUVEC was exhibited by selected strains of
S. gordonii
,
S. sanguis
,
S. mutans
,
S. mitis
, and
S. oralis
but only weakly by
S. salivarius
. Comparison of isogenic pairs of
S. gordonii
revealed a requirement for several surface proteins for maximum host cell invasion: glucosyltransferase, the sialic acid-binding protein Hsa, and the hydrophobicity/coaggregation proteins CshA and CshB. Deletion of genes for the antigen I/II adhesins, SspA and SspB, did not affect invasion. We hypothesize that peroxidogenesis and invasion of the cardiovascular endothelium by viridans group streptococci are integral events in the pathogenesis of infective endocarditis and atherosclerosis.
Aae, an Autotransporter Involved in Adhesion of
Actinobacillus actinomycetemcomitans
to Epithelial Cells
Authors: Rose John E. , Meyer Diane H. , Fives-Taylor Paula M.
Published: April 2003

The periodontal pathogen
Actinobacillus actinomycetemcomitans
possesses myriad virulence factors, among them the ability to adhere to and invade epithelial cells. Recent advances in the molecular manipulation of this pathogen and the sequencing of strain HK 1651 (
http://www.genome.ou.edu/act.html
) have facilitated examination of the genetics of its interaction with epithelial cells. The related gram-negative organism,
Haemophilus influenzae
, possesses autotransporter adhesins. A search of the sequence database of strain HK 1651 revealed a homologue with similarity in the pore-forming domain to that of the
H. influenzae
autotransporter, Hap.
A. actinomycetemcomitans
mutants deficient in the homologue, Aae, showed reduced binding to epithelial cells. A method for making
A. actinomycetemcomitans
SUNY 465 transiently resistant to spectinomycin was used with conjugation to generate an isogenic
aae
mutant. An allelic replacement mutant was created in the naturally transformable
A. actinomycetemcomitans
strain ATCC 29523. Lactoferrin, an important part of the innate host defense system, protects against bacterial infection by bactericidal and antiadhesion mechanisms. Lactoferrin in human milk removes or cleaves Hap and another autotransporter, an immunoglobulin A1 protease, from the surface of
H. influenzae
, thereby reducing their binding to epithelial cells. Human milk whey had similar effects on Aae from
A. actinomycetemcomitans
ATCC 29523 and its binding to epithelial cells; however, there was little effect on the binding of SUNY 465. A difference in the genetic structure of
aae
in the two strains, apparently due to the copy number of a 135-base repeated sequence, may be the cause of the differential action of lactoferrin.
aae
is the first
A. actinomycetemcomitans
gene involved in adhesion to epithelial cells to be identified.
Parasite-Specific Immunomodulatory Functions of Filarial Cystatin
Authors: Schierack Peter , Lucius Richard , Sonnenburg Bettina , Schilling Klaus , Hartmann Susanne
Published: April 2003

Cystatins of parasitic nematodes are well-described pathogenicity factors which contribute to downregulation of T-cell proliferation of their hosts and induce anti-inflammatory cytokine responses. We compared the immunomodulatory effects of two cystatins of the filarial nematodesOnchocerca volvulusandAcanthocheilonema viteaewith two homologous proteins of the free-living nematodeCaenorhabditis elegans.Like filarial cystatins, theC. eleganscystatins (rCysele1 and rCysele2) possessed domains relevant for inhibition of papain-like proteases and were biologically active inhibitors of human cathepsins B, L, and S. However, the inhibition of cathepsin B byC. eleganscystatin was much stronger.C. eleganscystatins lacked a domain involved in inhibition of legumain-like proteases that was present inO. volvuluscystatin. Filarial cystatins suppressed the proliferation of human peripheral blood mononuclear cells (PBMC) and murine spleen cells, while theC. eleganscystatins had this effect to a much lesser extent. Whereas filarial cystatins markedly increased the production of interleukin (IL)-10,C. eleganscystatins increased the production of IL-12 and gamma interferon (IFN-γ) by human PBMC. The cystatins of both the filariae andC. elegansinduced an upregulation of inducible nitric oxide by IFN-γ-stimulated murine macrophages. These data suggest that filarial cystatins but not theC. eleganscystatins downregulate proliferative responses of host cells due to characteristics which might reflect an adaptation of filariae to their parasitic life style.
Isolation of cDNAs Encoding Secreted and Transmembrane Proteins fromSchistosoma mansoniby a Signal Sequence Trap Method
Authors: Smyth Danielle , McManus Donald P. , Smout Michael J. , Laha Thewarach , Zhang Wenbao , Loukas Alex
Published: April 2003

Surface and secreted proteins of schistosomes orchestrate the basic physiologic requirements of a parasitic existence. These proteins are often exposed to host tissues during penetration, migration, feeding, and immune evasion, and they are obvious targets for control strategies. Signal sequence trap (SST) represents a novel approach that selects for cDNAs encoding secreted and surface proteins with N-terminal signal peptides, so we constructed a randomly primed adultSchistosoma mansonicDNA library fused to a signalless reporter gene encoding placental alkaline phosphatase. The library was used to transfect COS-7 cells, which were then assayed for the presence of reporter at the cell surface. EighteenS. mansonicDNA fragments were isolated and sequenced. Expression profiles of the novel clones were determined for different developmental stages; some transcripts were restricted to single-sex adult worms, while others were ubiquitously distributed. Most clones contained signal peptides or signal anchors as determined by the SignalP algorithm. Open reading frames (ORFs) were categorized as follows: (i) previously identifiedS. mansonicDNAs encoding proteins of known function; (ii) cDNAs encoding proteins of known function in other organisms but novel forSchistosoma; (iii)S. mansoniexpressed sequence tags (ESTs) of unknown function; and (iv) completely novel ORFs without homologues (including ESTs) from any phylum. Clones of particular interest included tetraspanins similar to human cell surface antigens, a protein kinase, and ORFs transcribed in the antisense orientation to previously characterizedS. mansonicDNAs. This is the first report describing the use of SST as a tool for identifying secreted proteins from any pathogenic organism.
Temporal Expression of Type III Secretion Genes ofChlamydia pneumoniae
Authors: Slepenkin Anatoly , Motin Vladimir , de la Maza Luis M. , Peterson Ellena M.
Published: April 2003

Chlamydia pneumoniaehas been shown to possess at least 13 genes that are homologous with other known type III secretion (TTS) systems. Upon infection of HEp-2 cells withC. pneumoniae, the expression of these genes was followed by reverse transcriptase PCR throughout the developmental cycle of this obligate intracellular pathogen. In addition, expression was analyzed whenC. pneumoniaewas grown in the presence of human gamma interferon (IFN-γ). ThegroEL-1,ompA, andomcBgenes were used as markers for the early, middle, and late stages of the developmental cycle, respectively, and the inhibition of expression of thefstKgene was used as a marker for the effect of IFN-γ on the maturation ofC. pneumoniae. In the absence of IFN-γ, the TTS genes were expressed as follows: early stage (1.5 to 8 h),yscC,yscS,yscL,yscJandlcrH-2; middle stage (by 12 to 18 h),lcrD,yscN, andyscR; and late stage (by 24 h),lcrE,sycE,lcrH-1, andyscT. Of the genes expressed early, thelcrH-2gene was detected the earliest, at 1.5 h. Expression of theyscUgene was not detected at any of the time points examined. Under the influence of IFN-γ, the cluster of TTS genes that were normally not expressed until the middle to late stages of the developmental cycle, namely,lcrD,lcrE, andsycE, as well aslcrH-1, were down-regulated, and expression could not be detected up to 48 h. In contrast, the expression of the other TTS genes appeared to be unchanged in the presence of IFN-γ. ThelcrH-1andlcrH-2genes differed from one another in both their temporal expression and response to IFN-γ. In other TTS systems, these genes code for proteins that function in regulation of effector protein synthesis as well as serve as chaperones for proteins that provide for the translocation of the effector proteins into the host cell. In summary, the expression pattern of the TTS genes ofC. pneumoniaeexamined suggests that they are temporally regulated throughout the developmental cycle. Furthermore, paralleling the inhibition of the maturation of the reticulate body to the elementary body, TTS genes expressed in the later stages of the cycle appear to be down-regulated when the organism is grown in the presence of IFN-γ.
Unexpected Similarities betweenBordetella aviumand Other Pathogenic Bordetellae
Authors: Spears Patricia A. , Temple Louise M. , Miyamoto David M. , Maskell Duncan J. , Orndorff Paul E.
Published: April 2003

Bordetella aviumcauses an upper respiratory tract disease (bordetellosis) in avian species. Commercially raised turkeys are particularly susceptible. Like other pathogenic members of the genusBordetella(B. pertussisandB. bronchiseptica) that infect mammals,B. aviumbinds preferentially to ciliated tracheal epithelial cells and produces similar signs of disease. These similarities prompted us to study bordetellosis in turkeys as a possible nonmammalian model for whooping cough, the exclusively human childhood disease caused byB. pertussis. One impediment to accepting such a host-pathogen model as relevant to the human situation is evidence suggesting thatB. aviumdoes not express a number of the factors known to be associated with virulence in the other twoBordetellaspecies. Nevertheless, with signature-tagged mutagenesis, four avirulent mutants that had lesions in genes orthologous to those associated with virulence inB. pertussisandB. bronchiseptica(bvgS,fhaB,fhaC, andfimC) were identified. None of the fourB. aviumgenes had been previously identified as encoding factors associated with virulence, and three of the insertions (infhaB,bvgS, andfimC) were in genes or gene clusters inferred as being absent or incomplete inB. avium, based upon the lack of DNA sequence similarities in hybridization studies and/or the lack of immunological cross-reactivity of the putative products. We further found that the genotypic arrangements of most of theB. aviumorthologues were very similar in all threeBordetellaspecies. In vitro tests, including hemagglutination, tracheal ring binding, and serum sensitivity, helped further define the phenotypes conferred by the mutations. Our findings strengthen the connection between the causative agents and the pathogenesis of bordetellosis in all hosts and may help explain the striking similarities of the histopathologic characteristics of this upper airway disease in avian and mammalian species.
RegR, a Global LacI/GalR Family Regulator, Modulates Virulence and Competence in
Streptococcus pneumoniae
Authors: Chapuy-Regaud Sabine , Ogunniyi A. David , Diallo Nicole , Huet Yvette , Desnottes Jean-François , Paton James C. , Escaich Sonia , Trombe Marie-Claude
Published: April 2003

The homolactic and catalase-deficient pathogen
Streptococcus pneumoniae
is not only tolerant to oxygen but requires the activity of its NADH oxidase, Nox, to develop optimal virulence and competence for genetic transformation. In this work, we show that the global regulator RegR is also involved in these traits. Genetic dissection revealed that RegR regulates competence and the expression of virulence factors, including hyaluronidase. In bacteria grown in vitro, RegR represses hyaluronidase. At neutral pH, it increases adherence to A549 epithelial cells, and at alkaline pH, it acts upstream of the CiaRH two-component signaling system to activate competence. These phenotypes are not associated with changes in antibiotic resistance, central metabolism, and carbohydrate utilization. Although the RegR
0
(where 0 indicates the loss of the protein) mutation is sufficient to attenuate experimental virulence of strain 23477 in mice, the introduction of an additional

hyl
0

(where 0 indicates the loss of function) mutation in the RegR
0
strain 23302 dramatically reduces its virulence. This indicates that residual virulence of the RegR
0
Hyl
+
derivative is due to hyaluronidase and supports the dual role of RegR in virulence. This LacI/GalR regulator, not essential for in vitro growth in rich media, is indeed involved in the adaptive response of the pneumococcus via its control of competence, adherence, and virulence.
Apoptotic Response of Chang Cells to Infection withPseudomonas aeruginosaStrains PAK and PAO-I: Molecular Ordering of the Apoptosis Signaling Cascade and Role of Type IV Pili
Authors: Jendrossek Verena , Fillon Sophie , Belka Claus , Müller Ilka , Puttkammer Beatrice , Lang Florian
Published: April 2003

Pseudomonas aeruginosais a gram-negative facultative opportunistic pathogen associated with severe infections in immunocompromised hosts and in patients with cystic fibrosis.P. aeruginosastrains show divergent pathogenicity in vivo and trigger apoptosis of and/or are internalized into human host cells. In the present study, we studied the molecular ordering of apoptosis signaling upon infection of human conjunctiva epithelial Chang cells withP. aeruginosaPAK as well as the role of bacterial pili in the response to the infection. Our results show that CD95 up-regulation is followed by early activation of caspase-8 and -3 and cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase. The data also demonstrate release of apoptosis inducing factor into the cytosol of infected cells. Induction of mitochondrial alterations, i.e., mitochondrial depolarization and release of cytochrome c, as well as cleavage of caspase-9, -7, and -1 occurred only at later time points. In addition, our results demonstrate that pili are required forP. aeruginosa-induced apoptosis of human epithelial cells. While the two piliatedP. aeruginosastrains, PAO-I and PAK, induced apoptosis of Chang cells within 3 h of infection, the pilus-deficientP. aeruginosamutants PAKΔpilAand PAKΔpilAΔallwere without effect. The pilus-deficient mutants failed to induce a significant up-regulation of CD95 on the cell surface and to trigger mitochondrial alterations or activation of caspase-8, -3, and -7. In addition, only the piliated wild-type strains induced caspase-1-mediated activation of interleukin-1β. Thus, pili are necessary for distinct infection-induced cellular responses of human epithelial cells.