bluuebunny/crossref_metadata_2025 huggingface dataset:
Calorimetric determination of vitrification time and heat capacity of a thermosetting polymer
Authors: Cassettari M. , Salvetti G. , Tombari E. , Veronesi S. , Johari G. P.
Published: April 2003

First observations of a decrease in heat capacity on isothermal vitrification of a thermosetting polymer are reported. The method developed for this study allows simultaneous measurements of both the heat capacity of a material and the enthalpy released as chemical reactions occur under virtually isothermal conditions. Control experiments have been done on a nonstoichiometric composition which does not vitrify and for which total enthalpy released can be measured. During isothermal curing, the heat capacity of a thermoset first increases slightly, undergoes an abrupt decrease in a narrow range of time, and thereafter slowly decreases. The abrupt decrease represents the transition of the liquid to a glassy solid, which is irreversible and which shifts to shorter times as the curing temperature is increased. This transition occurs at different extents of cure for different temperatures. The thermodynamic consequence of kinetic slowing on irreversible conversion of a molecular liquid to a macromolecule under isothermal conditions is similar to that observed on reversible clustering, as a result of decrease in the thermal energy on supercooling a molecular liquid. © 1993 John Wiley & Sons, Inc.
Conformation of the ground‐state dimer in poly(ethylene terephthalate)
Authors: Dodge Robert W. , Mattice Wayne L.
Published: April 2003

Absorbance, excitation, and emission measurements have been performed with methyl benzoate and five model compounds, C6H5COO (CH2)xOOCC6H5, x = 2–6. Under appropriate conditions, three of the model compounds (those with x = 3, 4, 5) show evidence for the formation of intramolecular ground‐state dimers. The model compound with x = 5 can form two types of dimers which emit with different energies. The model compound with x = 3 forms one of these dimers, and the model compound with x = 4 prefers the other ground‐state dimer. Molecular modeling of the dimers suggests that the two conformations of the ground‐state dimers differ in the orientation of the two CO bonds. In the one dimer these two bonds are nearly parallel, but in the other they make an angle of about 120°. © 1993 John Wiley & Sons, Inc.
The ratio of excimer to monomer emission in diesters of 1‐pyrenoic acid and five glycols
Authors: Mendicuti Francisco , Saiz Enrique , Mattice Wayne L.
Published: April 2003

The ratio of the intensities of excimer and monomer emission, ID/IM, has been measured for PyCOO(CH2)mOOCPy (m = 2–6, Py = 1‐pyrenyl) in solvents of different viscosity, η. These compounds are dimeric models for the corresponding polymers, selected to eliminate the influence of energy migration on the population of an excimer. The solvents are H(CH2)nOH, n = 1–7, and ethylene glycol. The values of ID/IM decrease as η increases, and the rate of this decrease is a function of m. Therefore the qualitative dependence of ID/IM on m is a function of η. In the limit as η → ∞, ID/IM is an odd–even function of m for m = 2–5, and the values at m = 5 and 6 are nearly identical. A rotational‐isomeric state analysis of the conformations of the diesters can rationalize the appearance of the odd–even effect. This result is insensitive to assumptions about the extent of overlap of the two pyrene ring systems that is required for the production of emission in the excimer band. © 1993 John Wiley & Sons, Inc.
Measurement of the interfacial tension between polyethersulfone and a thermotropic liquid‐crystalline copolyester
Authors: James S. G. , Donald A. M. , Miles I. S. , Mallach L. , Macdonald W. A.
Published: April 2003

Due to the increasing interest in forming blends of liquid‐crystalline polymers with conventional thermoplastics, it becomes important to determine the interfacial tension between two such polymers. A method for evaluating the interfacial tension between a thermotropic copolyester based on hydroxybenzoic and hydroxynaphthoic acid residues, and polyethersulfone is presented, based on the Fort and Patterson method. It is found that the value of the interfacial tension in the melt is much higher than is the case between conventional polymer pairs. It is suggested that this high value reflects an entropic effect due to the strong exclusion of the flexible coil polymer from the nematic melt. © 1993 John Wiley & Sons, Inc.
Stress experiences of parents with premature infants in a special care nursery
Authors: Lau Rosalind , Morse Carol A.
Published: April 2003

A controlled prospective longitudinal study was carried out to determine the stress experiences of parents whose premature infants were admitted to a tertiary‐based special care nursery in Melbourne, Australia. The control group consisted of parents of term infants matched on maternal age, parity and socioeconomic status. Sixty mothers and 59 fathers of premature infants and 60 mothers and fathers of term infants were followed from birth to 16 weeks after discharge home. A range of repeated self‐reports was carried out regarding feelings, moods, marital/partner relationships, availability and satisfaction with perceived social support and salivary markers of stress (cortisol and tribulin) were collected. Assessments occurred on five occasions with parents of premature infants and with parents of healthy term infants, data were collected on three occasions. Findings revealed that parents of premature infants reported higher subjective stress levels than parents of term infants within the first week of their infant's birth but returned lower objective markers of stress. There was no correlation between self‐reported stress and the biochemical markers of stress. The stress levels for parents of premature infants reduced over time. Issues for future research are discussed. Copyright © 2003 John Wiley & Sons, Ltd.
VMAT2 binding is elevated in dopa‐responsive dystonia: Visualizing empty vesicles by PET
Authors: De La Fuente‐Fernández Raúl , Furtado Sarah , Guttman Mark , Furukawa Yoshiaki , Lee Chong S. , Calne Donald B. , Ruth Thomas J. , Stoessl A. Jon
Published: April 2003

Dopa‐responsive dystonia (DRD) is a lifelong disorder in which dopamine deficiency is not associated with neuronal loss and therefore it is an ideal human model for investigating the compensatory changes that occur in response to this biochemical abnormality. Using positron emission tomography (PET), we examined the (±)‐α‐[11C]dihydrotetrabenazine ([11C]DTBZ) binding potential of untreated DRD patients and normal controls. Two other PET markers of presynaptic nigrostriatal function, d‐threo‐[11C]methylphenidate ([11C]MP) and 6‐[18F]fluoro‐L‐dopa ([18F]‐dopa), and [11C]raclopride were also used in the study. We found increased [11C]DTBZ binding potential in the striatum of DRD patients. By contrast, no significant changes were detected in either [11C]MP binding potential or [18F]‐dopa uptake rate constant. In addition, we found evidence for increased dopamine turnover in one DRD patient by examining changes in [11C]raclopride binding potential in relation to levodopa treatment. We propose that the increase in [11C]DTBZ binding likely reflects the dramatic decrease in the intravesicular concentration of dopamine that occurs in DRD; upregulation of vesicular monoamine transporter type 2 (VMAT2) expression may also contribute. Our findings suggest that the striatal expression of VMAT2 (as estimated by [11C]DTBZ binding) is not coregulated with dopamine synthesis. This is in keeping with a role for VMAT2 in other cellular processes (i.e., sequestration and release from the cell of potential toxic products), in addition to its importance for the quantal release of monoamines. Synapse 49:20–28, 2003. © 2003 Wiley‐Liss, Inc.
β‐Funaltrexamine, a gauge for the recognition site of wildtype and mutant H297Q μ‐opioid receptors
Authors: Spivak Charles E. , Beglan Carol L. , Zöllner Christian , Surratt Christopher K.
Published: April 2003

The antagonist β‐funaltrexamine (β‐FNA), known to bind covalently to μ‐opioid receptors by a two‐step, doubly discriminating sequence, was used as a sensitive gauge to compare wildtype to mutant H297Q μ‐opioid receptors. We addressed whether this mutation, which enhances the intrinsic activities of alkaloid μ‐receptor agents, affects both the reversible and covalent phases of β‐FNA binding. Such altered binding serves as a reporter for the dimensions and topography of the receptor's recognition site. Using the voltage‐clamped Xenopus oocyte expression system with coexpressed GIRK potassium channels, we found that β‐FNA blocked the wildtype and mutant H297Q receptors both reversibly and irreversibly, indicating overall conserved tertiary structure in the mutant. The mutant H297Q receptor, however, was more resistant to both phases of blockade, indicating some disturbance of the mutant H297Q receptor recognition site. β‐FNA acted as a partial agonist at the mutant H297Q receptor expressed in both oocytes, as measured by the activation of GIRK channels, and in COS‐7 cells assayed by GTPγ35S binding. β‐FNA showed no activity at the wildtype receptor expressed in oocytes, but surprisingly induced binding of GTPγ35S in transfected COS‐7 cells. Thus, the topography of the mutant H297Q receptor recognition site is sufficiently conserved to allow the selective binding of β‐FNA, but the decrease in binding affinity and increase in efficacy in oocytes demonstrates clear differences from the wildtype receptor. Synapse 49:55–60, 2003. © 2003 Wiley‐Liss, Inc.
Requirement of a critical period of GABAergic receptor blockade for induction of a cAMP‐mediated long‐term depression at CA3‐CA1 synapses
Authors: Yu Tzu‐Ping , Lester Henry A. , Davidson Norman
Published: April 2003

Previous reports show that bath application of the adenosine 3′ : 5′‐cyclic monophosphate (cAMP) analog, Sp‐cAMPS, induces a protein kinase A (PKA)‐dependent and protein synthesis‐dependent long‐term potentiation (LTP) at hippocampal CA3‐CA1 synapses. Recently, we reported a novel form of long‐term depression (LTD) induced by concurrent application of Sp‐cAMPS and picrotoxin, the γ‐aminobutyric acid type A (GABAA) receptor antagonist. In the present study, we further investigated the mechanisms underlying such cAMP‐mediated LTD. Synaptically connected CA3 and CA1 cells of hippocampal slice cultures were impaled by sharp electrodes. Excitatory postsynaptic potentials recorded from a CA1 pyramidal cell were evoked by single action potentials in a CA3 cell. Picrotoxin was applied to slices at various time points after Sp‐cAMPS was perfused. We found that Sp‐cAMPS‐induced potentiation could be converted to depression when picrotoxin was applied within 30 min after perfusion of Sp‐cAMPS. Picrotoxin applied 1 h after perfusion of Sp‐cAMPS had no effect on Sp‐cAMPS‐induced synaptic potentiation. Once LTP was induced by Sp‐cAMPS and expressed for 1 h, the subsequent application of Sp‐cAMPS and picrotoxin produced no new changes in synaptic strength. Also, once LTD was induced and expressed for 1 h, subsequent Sp‐cAMPS produced no new changes in synaptic strength. These findings suggest that a synapse is committed irreversibly to cAMP‐mediated LTP or LTD during a critical period and that later signals cannot interconvert these two fates. Synapse 49:12–19, 2003. © 2003 Wiley‐Liss, Inc.
Behavioral/neurophysiological investigation of effects of combining a quinolinic acid entopeduncular lesion with a fetal mesencephalic tissue transplant in striatum of the 6‐OHDA hemilesioned rat
Authors: Olds M.E. , Jacques D.B. , Kpoyov O.
Published: April 2003

Behavioral and electrophysiological methods were used to investigate the effects of combining a unilateral quinolinic acid lesion of the entopeduncular nucleus (QA/EP) with a striatal transplant of fetal ventral mesencephalic tissue in the 6‐hydroxydopamine (6‐OHDA) hemilesioned rat model for Parkinson's disease. The subjects were 6‐OHDA‐lesioned rats selected because their response to amphetamine treatment was a strongly biased rotation toward the side of the 6‐OHDA lesion in the substantia nigra, at the expense of other evoked behaviors associated with amphetamine. Two experiments were performed. In the first, the motor effects of the QA/EP lesion alone and of the combination of QA/EP lesion with striatal transplant were determined by measuring six aspects of the motor response. In the second the electrophysiological effects of the two interventions on the responses of neurons in the subthalamic nucleus to amphetamine and apomorphine were determined in the 6‐OHDA‐lesioned rats. The results of the first experiment show that the QA/EP lesion by itself produced an attenuation of the rotation response and, simultaneously, an increase of oral stereotypy. They also show that the combination of QA/EP lesion with striatal transplant was more effective than the single intervention, inducing more attenuation of rotation and more oral stereotypy. The results of the second experiment show that the responses of subthalamic neurons to amphetamine in the behaving hemiparkinsonian rat with combined QA/EP lesion and transplant were larger than the responses in the hemiparkinsonian rat with the QA/EP lesion alone. However, even these larger responses in the rats with combined intervention were not as large as those recorded at the same time in the subthalamus in the opposite, intact, hemisphere. The results of the two experiments, both of which show enhanced motor and neuronal sensitivity to amphetamine after the combined intervention, suggest that such a multiple approach might prove more beneficial than a one‐site intervention targeting either the EP or the striatum. Synapse 49:1–11, 2003. © 2003 Wiley‐Liss, Inc.
Role of epinephrine stimulation of CNS α1‐adrenoceptors in motor activity in mice
Authors: Stone Eric A. , Grunewald Gary L. , Lin Yan , Ahsan Rashedul , Rosengarten Helen , Kramer H. Kenneth , Quartermain David
Published: April 2003

The role of brain epinephrine (EPI) in the regulation of motor activity and movement in mice was examined. Blockade of EPI synthesis with i.p. 2,3‐dichloro‐α‐methylbenzylamine (DCMB) or LY134046 was found to produce marked behavioral inactivity which could be significantly reversed by intraventricular injection of EPI and by three other α1‐adrenoceptor agonists, norepinephrine (NE), 6‐fluoronorepinephrine (6FNE), and phenylephrine (PE), as well as by serotonin (5HT). EPI had the largest effect of these agonists and also was the only one that reversed nondrug‐induced inactivity of mice in their home cages during the light phase. The effects of EPI were blocked by coinfusion of an α1‐adrenoceptor antagonist (terazosin) but not of an α2‐(atipamezole) or β1 (betaxolol)‐blocker. The rank order of maximal behavioral responses to EPI, 6FNE, and PE in DCMB‐treated mice was the same as the rank order of their maximal stimulation of hydrolysis of phosphatidylinositol at cloned α1B‐adrenoceptors in cell culture. On the basis of the above findings and of the central distributions of adrenergic neurons and α1‐adrenoceptors, the existence of a central EPI‐innervated α1‐adrenergic receptor system is postulated which serves to coexcite or enhance signaling in several monoaminergic brain regions involved in movement and motor activity. Synapse 49:67–76, 2003. © 2003 Wiley‐Liss, Inc.
Reliability analysis of wind response of flexibly supported tall structures
Authors: Halabian A. M. , El Naggar M. H. , Vickery B. J.
Published: April 2003

Statistics and probabilistic analyses and risk assessments can be very useful decision‐making tools when dealing with structural–geotechnical problems. Wind loads, dynamic properties of soil underneath the structure and material characteristics of the structure are important factors that affect the wind action on the structure and consequently the structural wind‐induced response. Uncertainties in the estimation of these factors as a result of human error or inherent variability are at the forefront for the use of reliability approaches to evaluate the risk of failure during the service period. In the present study, probabilistic base force analyses for tall structures are performed. The substructure approach in which the soil supporting the foundation is modeled by the foundation compliances as functions of soil shear wave velocity is used to account for the soil–structure interaction efficiently. A three‐variable probabilistic approach is used to account for the uncertainties in shear wave velocity of the soil underneath the foundation, the concrete strength and the design wind speed on the calculated response and the base forces. The second moment approximation using Taylor series expansion is used to perform the probabilistic analyses of the base cross‐section design and resistant forces of a free‐standing tower. The first‐order reliability method is used to examine the failure probability and the contribution to the total uncertainty. The results show that the dynamic response of the tower increases as soil shear wave velocity decreases. For the range of soil shear wave velocity encountered in practice, the base forces of the structure may increase by up to 20% as a result of the foundation flexibility. For the limit state considered in this study, it was found that the reliability index decreases by up to 15% and the probability of failure increases by up to one order of magnitude as a result of the soil–structure interaction effect. Copyright © 2003 John Wiley & Sons, Ltd.
Optimal compensation of differential column shortening in high‐rise buildings
Authors: Park H. S.
Published: April 2003

To avoid unexpected damage in structural and nonstructural elements, differential shortening between vertical members resulting from differing stress levels, loading histories, volume‐to‐surface ratios and other factors in a high‐rise building must be properly considered in the design process. While research activity has been reported in the literature on the development of estimation algorithms or prediction procedures for the elastic and inelastic shortenings of vertical members, no algorithms or systematic methods for the compensation of the differential shortenings have been reported. In this paper, a compensation method for the differential column shortening in a high‐rise building is formulated into an optimization problem. A simulated annealing algorithm is used to find optimal solutions. The proposed method is applied to the compensation of the differential shortening of the vertical members in two high‐rise buildings, including one verifying example of a 70‐storey building and a practical example of a 63‐storey building. As demonstrated in the examples, the differential shortenings of the examples are effectively controlled by the optimal compensation method. Copyright © 2003 John Wiley & Sons, Ltd.
Field measurements of wind effects on the tallest building in Hong Kong
Authors: Li Q. S. , Xiao Y. Q. , Wong C. K. , Jeary A. P.
Published: April 2003

Central Plaza has a height of approximately 374 m and is 78‐storeys tall. It is the tallest structure in Hong Kong and was the highest reinforced‐concrete building in the world when it was built several years ago. This paper describes some results obtained from the full‐scale measurements of wind effects on Central Plaza. The field data such as wind speed, wind direction and acceleration responses have been measured during the close passage of several typhoons in recent years. Detailed analysis of the field data is conducted to investigate wind effects on the tall building. The full‐scale measurements are compared with the wind tunnel results. The amplitude‐dependent characteristics of damping that were obtained by using the random decrement technique are investigated on the basis of the field measurements. Copyright © 2003 John Wiley & Sons, Ltd.
The deployment of IPv6 in an IPv4 world and transition strategies
Authors: Bouras C. , Karaliotas A. , Ganos P.
Published: April 2003

It is stated that the new version of the IP protocol, IPv6, is the answer to the majority of the problems that were raised during the enormous expansion of the Internet and its transformation to a global communication platform with commercial scope. At the same time it is also stated that IPv6 dominance will not be easy and there will be a period when the two versions of the protocol will co‐exist. In this paper we describe some of the main transition mechanisms that can be deployed in order to facilitate the transition process to the new version of the IP protocol. Furthermore, the presented mechanisms are discussed regarding their usability, usefulness and manageability. Describes the way some of these mechanisms were applied to the Greek Research & Technology Network (GRNET).
Web portal for multicast delivery management
Authors: Mannaert H. , De Gruyter B. , Adriaenssens P.
Published: April 2003

In this paper, a Web portal is presented for multicast communication management, providing fully automatic service management with integrated provisioning of hardware equipment. The portal is based on an open and configurable object‐oriented framework, that allows self‐provisioning by the user and the seamless integration with all types of multicast application software. As its topological structure is ideally suited for multicasting, and it allows the accurate control of the transmission bandwidth, the portal focuses currently on satellite as a delivery medium. The software architecture, the implementation, and the application usage of the Web portal for multicast delivery are described.
Security for IP multimedia services in the 3GPP third generation mobile system
Authors: Horn G. , Kröselberg D. , Müller K.
Published: April 2003

This paper surveys the security architecture of the IP multimedia core network subsystem (IMS) of the third generation mobile system which is known in Europe as UMTS. The main features of the security architecture include user specific features protecting the access of the IMS user, such as authentication and key agreement when a user registers and integrity protection of IMS access signalling, but also features for the user, independent protection of SIP signalling in the IMS core network. Authenticated registration is given an in‐depth treatment.
eCX: a secure infrastructure for e‐course delivery
Authors: Yau Joe C.K. , Hui Lucas C.K. , Cheung Bruce , Yiu S.M.
Published: April 2003

Online education has emerged as one of the major channels for dissemination of learning materials. As more and more organizations offer online distance learning courses, the security concerns of these online education systems become more and more critical, especially when the organizations rely on the registration fees of students to maintain the smooth running of the courses. Provides a mechanism, the Secure e‐Course eXchange (eCX), to protect the learning material from unauthorized dissemination, and shows how this mechanism can be integrated in the operation model of online learning course providers. The design of eCX is general enough to fit two operating models, namely the Institutional Server Model and the Corporate Server Model.
Closed conference signalling using the session initiation protocol
Authors: Miladinovic Igor , Stadler Johannes
Published: April 2003

In a closed conference the identity of all conference participants is known by others and all participants are notified when a new user joins the conference. The Session Initiation Protocol (SIP) supports signalling of several conference models, but not of closed conferences. Introduces an extension of SIP for closed multiparty conferences. The extension expands SIP for the functionality for discovery of participant identities in a conference. Furthermore, it ensures that each conference participant is notified before a new participant joins. Verifies this extension by applying it to two SIP conference models – conference with conference server and full‐mesh conference. Ends with an analysis of additional signalling traffic that this extension generates.
Effects of Nitric Oxide onPseudomonas aeruginosaInfection of Epithelial Cells from a Human Respiratory Cell Line Derived from a Patient with Cystic Fibrosis
Authors: Darling Katharine E. A. , Evans Thomas J.
Published: April 2003

Cystic fibrosis (CF) is characterized by airway inflammation and chronic bacterial lung infection, most commonly withPseudomonas aeruginosa, an opportunistic human pathogen. Despite the persistent airway inflammation observed in patients with CF, although phagocyte inducible nitric oxide synthase (iNOS) production is upregulated, expression of iNOS in the respiratory epithelium is markedly reduced. Given the antimicrobial action of NO, this may contribute to the chronic airway infection of this disease. To define the role of epithelium-derived NO in airway defense againstP. aeruginosa, we infected differentiated human bronchial epithelial cells derived from a patient with CF (CFBE41o- cells) with different strains of this pathogen at low multiplicities of infection. Using cells transfected with human iNOS cDNA, we studied the effect of NO onP. aeruginosareplication, adherence, and internalization.P. aeruginosaadherence to iNOS-expressing cells was reduced by 44 to 72% (P= 0.02) compared with control values. AbsoluteP. aeruginosauptake into these cells was reduced by 44%, but uptake expressed as a percentage of adherent bacteria did not differ from the control uptake. Survival ofP. aeruginosawithin iNOS-expressing cells was reduced at late times postinfection (P= 0.034). NO production did not alter host cell viability. NO production reducedP. aeruginosaadherence to human bronchial epithelial cells and enhanced killing of internalized bacteria, suggesting that a lack of epithelial iNOS in patients with CF may contribute toP. aeruginosainfection and colonization.
Failure of Surface Ring Mutant Strains ofHelicobacter mustelaeTo Persistently Infect the Ferret Stomach
Authors: Patterson M. M. , O'Toole P. W. , Forester N. T. , Noonan B. , Trust T. J. , Xu S. , Taylor N. S. , Marini R. P. , Ihrig M. M. , Fox J. G.
Published: April 2003

Helicobacter mustelae, the gastric pathogen of ferrets, produces an array of surface ring structures which have not been described for any other member of the genusHelicobacter, includingH. pylori. The unique ring structures are composed of a protein named Hsr. To investigate whether the Hsr rings are important for colonization of the ferret stomach, ferrets specific pathogen free forH. mustelaewere inoculated with an Hsr-deficient mutant strain or the wild-typeH. mustelaestrain. Quantitative cultures from antral biopsy specimens obtained at 3, 6, and 9 weeks postinoculation demonstrated no significant difference in the levels of bacteria in the ferrets that received the Hsr-negative strain and the ferrets infected with the parent strain. However, when the ferrets were biopsied at 12 and 15 weeks and necropsied at 18 weeks after infection, the levels of bacteria of the Hsr-negative strain in the stomach antrum were significantly reduced. This decline contrasted the robust antral colonization by the wild-type strain. The Hsr-negative strain did not efficiently colonize the gastric body of the study ferrets. Histological examination at 18 weeks postinoculation revealed minimal gastric inflammation in the animals that received the mutantH. mustelaestrain, a finding consistent with its waning infection status, whereas lesions characteristic of helicobacter infection were present in ferrets infected with the wild-type strain. Scant colonization by the Hsr-negativeH. mustelaestrain at the end of the 18-week study, despite initial successful colonization, indicates an inability of the mutant to persist, perhaps due to a specific host response.
lvgA, a NovelLegionella pneumophilaVirulence Factor
Authors: Edelstein Paul H. , Hu Baofeng , Higa Futoshi , Edelstein Martha A. C.
Published: April 2003

Several novelLegionella pneumophilavirulence genes were previously discovered by use of signature-tagged mutagenesis (P. H. Edelstein, M. A. Edelstein, F. Higa, and S. Falkow, Proc. Natl. Acad. Sci. 96:8190-8195, 1999). One of these mutants appeared to be defective in multiplication in guinea pig lungs and spleens, yet it multiplies normally in guinea pig alveolar macrophages. Here we report further characterization of the mutated gene and its protein and the virulence role of the gene. The complete sequence of the gene, now calledlvgA, is 627 bp long, and its protein product is approximately 27 kDa in size.lvgAwas present in all 50 strains ofL. pneumophilatested. No significant nucleic acid or protein homology was found in the GenBank database for the gene, nor were any distinctive motifs discovered in a search of other databases. The expression of both DotA and IcmX in thelvgAmutant was normal. Subcellular fractionation studies localized LvgA to the outer membrane fraction, and protease digestion studies suggested that at least some of the protein is surface expressed. No change in bacterial lipopolysaccharide composition or reactivity to serogroup-specific antisera was detected in the mutant. Growth competition studies with alveolar macrophages showed that the mutant was outcompeted by its parent 3-fold in 24 h and 24-fold in 48 h, in contrast to what was observed with the null phenotype in parallel testing with alveolar macrophages or with the A549 alveolar epithelial cell line. This macrophage defect of the mutant bacterium was due to slower growth, as the mutant invaded alveolar macrophages normally. Electron microscopy showed that the mutant bacterium resided in a ribosome-studded phagosome in alveolar macrophages, with no distinction from its parent. ThelvgAmutant was outcompeted by its parent about sixfold in guinea pig lungs and spleens; prolonged observation of infected animals showed no late-onset virulence of the mutant. Transcomplementation of the mutant restored the parental phenotype in guinea pigs. ThelvgAmutant was twofold more susceptible to killing by human β-defensin 2 but not to killing by other cationic peptides, serum complement, or polymorphonuclear neutrophils.lvgAis a novel virulence gene that is responsible for pleiotropic functions involving both extracellular and intracellular bacterial resistance mechanisms.
Use of the
Galleria mellonella
Caterpillar as a Model Host To Study the Role of the Type III Secretion System in
Pseudomonas aeruginosa
Pathogenesis
Authors: Miyata Sachiko , Casey Monika , Frank Dara W. , Ausubel Frederick M. , Drenkard Eliana
Published: April 2003

Nonvertebrate model hosts represent valuable tools for the study of host-pathogen interactions because they facilitate the identification of bacterial virulence factors and allow the discovery of novel components involved in host innate immune responses. In this report, we determined that the greater wax moth caterpillar
Galleria mellonella
is a convenient nonmammalian model host for study of the role of the type III secretion system (TTSS) in
Pseudomonas aeruginosa
pathogenesis. Based on the observation that a mutation in the TTSS
pscD
gene of
P. aeruginosa
strain PA14 resulted in a highly attenuated virulence phenotype in
G. mellonella
, we examined the roles of the four known effector proteins of
P. aeruginosa
(ExoS, ExoT, ExoU, and ExoY) in wax moth killing. We determined that in
P. aeruginosa
strain PA14, only ExoT and ExoU play a significant role in
G. mellonella
killing. Strain PA14 lacks the coding sequence for the ExoS effector protein and does not seem to express ExoY. Moreover, using Δ
exoU
Δ
exoY
, Δ
exoT
Δ
exoY
, and Δ
exoT
Δ
exoU
double mutants, we determined that individual translocation of either ExoT or ExoU is sufficient to obtain nearly wild-type levels of
G. mellonella
killing. On the other hand, data obtained with a Δ
exoT
Δ
exoU
Δ
exoY
triple mutant and a Δ
pscD
mutant suggested that additional, as-yet-unidentified
P. aeruginosa
components of type III secretion are involved in virulence in
G. mellonella
. A high level of correlation between the results obtained in the
G. mellonella
model and the results of cytopathology assays performed with a mammalian tissue culture system validated the use of
G. mellonella
for the study of the
P. aeruginosa
TTSS.
Differences in Gamma Interferon Production Induced by Listeriolysin O and Ivanolysin O Result in Different Levels of Protective Immunity in Mice Infected with
Listeria monocytogenes
and
Listeria ivanovii
Authors: Kimoto Terumi , Kawamura Ikuo , Kohda Chikara , Nomura Takamasa , Tsuchiya Kohsuke , Ito Yutaka , Watanabe Isao , Kaku Taijin , Setianingrum Endang , Mitsuyama Masao
Published: April 2003

Two pathogenic species in the genus
Listeria, Listeria monocytogenes
and
Listeria ivanovii
, are characterized by the production of hemolysins belonging to cholesterol-dependent cytolysins, listeriolysin O (LLO) and ivanolysin O (ILO), respectively. LLO, produced by
L. monocytogenes
, is able to induce gamma interferon (IFN-γ) production and contributes to the generation of Th1-dependent protective immunity. On the other hand, nothing is known about the role of ILO, produced by
L. ivanovii
, in this regard. In this study, we immunized mice with 0.1 50% lethal dose (LD
50
) of
L. monocytogenes
and
L. ivanovii.
Protective immunity against a challenge with 10 LD
50
was generated in mice infected with
L. monocytogenes
, whereas
L. ivanovii
infection did not induce protection. After immunization, the level of IFN-γ in serum samples was increased in mice given
L. monocytogenes
but not in those given
L. ivanovii.
To determine the IFN-γ-inducing activity of cytolysins, recombinant protein was constructed. Recombinant ILO exhibited significantly lower IFN-γ-inducing activity than LLO. By comparing the IFN-γ-inducing activity of a chimera incorporating LLO and ILO, it was found that domains 1 to 3 of LLO were critical for IFN-γ-inducing activity while the counterpart in ILO was unable to induce cytokine production. These results suggested that the weak ability of ILO to induce IFN-γ production is responsible for the failure of
L. ivanovii
to generate effective protective immunity.
Gamma Interferon Production by Hepatic NK T Cells duringEscherichia coliInfection Is Resistant to the Inhibitory Effects of Oxidative Stress
Authors: Zhang Guochi , Nichols Robert Dru , Taniguchi Masaru , Nakayama Toshinori , Parmely Michael J.
Published: April 2003

The reductive-oxidative status of tissues regulates the expression of many inflammatory genes that are induced during gram-negative bacterial infections. The cytokine gamma interferon (IFN-γ) is a potent stimulus for host inflammatory gene expression, and oxidative stress has been shown to inhibit its production in mice challenged withEscherichia colibacteria. The objective of the present study was to characterize the cells that produced IFN-γ in a mouse bacterial peritonitis model and determine the effects of oxidative stress on their activation. The liver contained large numbers of IFN-γ-expressing lymphocytes following challenge with viableE. colibacteria. The surface phenotypes of IFN-γ-expressing hepatic lymphocytes were those of natural killer (NK) cells (NK1.1+CD3−), conventional T cells (NK1.1−CD3+), and NK T cells (NK1.1+CD3+). Treating mice with diethyl maleate to deplete tissue thiols significantly impaired IFN-γ production by NK cells, conventional T cells, and CD1d-restricted NK T cells in response toE. colichallenge. However, IFN-γ expression by a subset of NK T cells, which did not bind α-galactosylceramide-CD1d tetramers, was resistant to the inhibitory effects of tissue oxidative stress. Stress-resistant IFN-γ-expressing cells were also predominantly CD8+and bore γδ T-cell antigen receptors. The residual IFN-γ response by NK T cells may explain previous reports of hepatic gene expression following gram-negative bacterial challenge in thiol-depleted mice. The finding also demonstrates that innate immune cells differ significantly in their responses to altered tissue redox status.
Mutations in thelspA1andlspA2Genes ofHaemophilus ducreyiAffect the Virulence of This Pathogen in an Animal Model System
Authors: Ward Christine K. , Latimer Jo L. , Nika Joseph , Vakevainen Merja , Mock Jason R. , Deng Kaiping , Blick Robert J. , Hansen Eric J.
Published: April 2003

Haemophilus ducreyi35000HP contains two genes,lspA1andlspA2, whose predicted protein products have molecular weights of 456,000 and 543,000, respectively (C. K. Ward, S. R. Lumbley, J. L. Latimer, L. D. Cope, and E. J. Hansen, J. Bacteriol. 180:6013-6022, 1998). We have constructed threeH. ducreyi35000HP mutants containing antibiotic resistance cartridges in one or both of thelspA1andlspA2open reading frames. Western blot analysis using LspA1- and LspA2-specific monoclonal antibodies indicated that the wild-type parent strain 35000HP expressed LspA1 protein that was readily detectable in culture supernatant fluid together with a barely detectable amount of LspA2 protein. ThelspA2mutant 35000HP.2 expressed LspA1 protein that was detectable in culture supernatant fluid and no LspA2 protein. In contrast, theH. ducreyi lspA1mutant 35000HP.1, which did not express the LspA1 protein, expressed a greater quantity of the LspA2 protein than did the wild-type parent strain. ThelspA1 lspA2double mutant 35000HP.12 expressed neither LspA1 nor LspA2. The three mutant strains adhered to human foreskin fibroblasts and to a human keratinocyte cell line in vitro at a level that was not significantly different from that of the wild-type strain 35000HP. Lack of expression of the LspA1 protein by both thelspA1mutant and thelspA1 lspA2double mutant was associated with an increased tendency to autoagglutinate. When evaluated in the temperature-dependent rabbit model for chancroid, thelspA1 lspA2double mutant was substantially less virulent than the wild-type strain 35000HP. The results of these studies indicated thatH. ducreyirequires both the LspA1 and LspA2 proteins to be fully virulent in this animal model for experimental chancroid.
Epitope-Specific Humoral Immunity to
Plasmodium vivax
Duffy Binding Protein
Authors: Xainli Jia , Cole-Tobian Jennifer L. , Baisor Moses , Kastens Will , Bockarie Moses , Yazdani Syed Shams , Chitnis Chetan E. , Adams John H. , King Christopher L.
Published: April 2003

Erythrocyte invasion by
Plasmodium vivax
is completely dependent on binding to the Duffy blood group antigen by the parasite Duffy binding protein (DBP). The receptor-binding domain of this protein lies within a cysteine-rich region referred to as region II (DBPII). To examine whether antibody responses to DBP correlate with age-acquired immunity to
P. vivax
, antibodies to recombinant DBP (rDBP) were measured in 551 individuals residing in a village endemic for
P. vivax
in Papua New Guinea, and linear epitopes mapped in the critical binding region of DBPII. Antibody levels to rDBP
II
increased with age. Four dominant linear epitopes were identified, and the number of linear epitopes recognized by semiimmune individuals increased with age, suggesting greater recognition with repeated infection. Some individuals had antibodies to rDBP
II
but not to the linear epitopes, indicating the presence of conformational epitopes. This occurred in younger individuals or subjects acutely infected for the first time with
P. vivax
, indicating that repeated infection is required for recognition of linear epitopes. All four dominant B-cell epitopes contained polymorphic residues, three of which showed variant-specific serologic responses in over 10% of subjects examined. In conclusion, these results demonstrate age-dependent and variant-specific antibody responses to DBPII and implicate this molecule in partial acquired immunity to
P. vivax
in populations in endemic areas.
Kinetics of Antibody Response toEhrlichia canisImmunoreactive Proteins
Authors: McBride Jere W. , Corstvet Richard E. , Gaunt Steven D. , Boudreaux Charles , Guedry Thaya , Walker David H.
Published: April 2003

Immunoreactive proteins ofEhrlichia canisandEhrlichia chaffeensisthat have been characterized include a family of 28-kDa major outer membrane proteins (p28) and two large antigenically divergent surface glycoprotein orthologs. We previously demonstrated that recombinantE. canisp28 and the 140- and 200-kDa glycoproteins gp140 and gp200, respectively, react strongly with serum antibodies from suspect canine ehrlichiosis cases that were positive forE. canisby immunofluorescent antibody test and in various phases of acute or chronic infection (J. Clin. Microbiol. 39:315-322, 2001). The kinetics of the antibody response to these potentially important vaccine and immunodiagnostic candidates is not known. Acute-phase serum antibody responses to whole-cellE. canislysates and recombinant p28, gp140, and gp200 were monitored for 6 weeks in dogs experimentally infected withE. canis. Irrespective of the inoculation route, a T-helper 1-type response was elicited toE. canisantigens consisting of immunoglobulin G2 antibodies exclusively in both acute and convalescent phases in most dogs. Analysis of immuoreactive antigens for peak intensity and relative quantity identified major immunoreactiveE. canisantigens recognized early in the infection as the 19-, 37-, 75-, and 140-kDa proteins. Later in infection, additional major immunoreactiveE. canisproteins were identified, including the 28-, 47-, and 95-kDa proteins and the recently identified 200-kDa glycoprotein. All dogs had developed antibody against the recombinant gp140, gp200, and p28 in the convalescent phase. Immunoreactivity and antibody response kinetics suggest that major immunoreactive proteins identified are immunodominant, but early recognition suggests increased dominance by some antigens.
Identification of aTreponema pallidumLaminin-Binding Protein
Authors: Cameron Caroline E.
Published: April 2003

Host extracellular matrix (ECM) components represent ideal microbial adhesion targets that many pathogens use for colonization of tissues and initiation of infection. This study investigated the interaction of the spirocheteTreponema pallidumwith the ECM component laminin. To identify candidate laminin-binding adhesins, theT. pallidumgenome was analyzed to predict open reading frames that encode putative outer membrane proteins, as these proteins interact directly with host ECM components. Subsequent recombinant expression of these proteins and analysis of their laminin-binding potential identified one protein, Tp0751, that demonstrated specific attachment to laminin. Tp0751 attached to laminin in a dose-dependent, saturable manner but did not attach to the ECM component collagen type I or IV or to the negative control proteins fetuin or bovine serum albumin. Sodium metaperiodate treatment of laminin reduced the Tp0751-laminin interaction in a concentration-dependent manner, suggesting that oligosaccharides play a role in this interaction. In addition, Tp0751-specific antibodies were detected in serum samples collected from both experimental and natural syphilis infections, indicating that Tp0751 is expressed in vivo during the course of infection. Collectively, these experiments identified Tp0751 as a laminin-binding protein that is expressed during infection and may be involved in attachment ofT. pallidumto host tissues.
Development of an Interleukin-12-Deficient Mouse Model That Is Permissive for Colonization by a Motile KE26695 Strain of
Helicobacter pylori
Authors: Hoffman Paul S. , Vats Neeraj , Hutchison Donna , Butler Jared , Chisholm Kenneth , Sisson Gary , Raudonikiene Ausra , Marshall Jean S. , Veldhuyzen van Zanten Sander J. O.
Published: April 2003

The identification of genes associated with colonization and persistence of
Helicobacter pylori
in the gastric mucosa has been limited by the lack of robust animal models that support infection by strains whose genomes have been completely sequenced. Here we report that an interleukin-12 (IL-12)-deficient mouse (IL-12
−/−
p40 subunit knockout in C57BL/6 mouse) is permissive for infection by a motile variant (KE88-3887) of The Institute For Genomic Research-sequenced strain (KE26695) of
H. pylori
. The IL-12-deficient mouse was also more permissive for colonization by the mouse-colonizing Sydney 1 strain of
H. pylori
than were wild-type C57BL/6 mice. Differences in colonization efficiency were demonstrated by mouse challenge with SS1 strains containing loss-of-function mutations in two genes (
hspR
and
hrcA
), whose products negatively regulate several heat shock genes. At 5 weeks postinfection, double-knockout mutants (SS1
hspR hrcA
) efficiently colonized IL-12-deficient mice (5 of 5 animals compared to 4 of 10 for C57BL6 mice) and bacterial counts were higher in stomachs of IL-12-deficient mice (10
6
versus 10
5
CFU/g of stomach, respectively). IL-12-deficient mice were efficiently colonized by KE88-3887 (29 of 30), but not by nonmotile KE26695, and bacterial numbers (10
4
to 10
5
CFU/g of stomach) were unchanged over an 8-week period postinfection. In contrast, C57BL/6 mice were inefficiently colonized by KE88-3887 (8 of 20 animals with bacterial loads at the limit of detection, ∼10
3
CFU/g), and infection did not persist much beyond 5 weeks. Cytokine responses (tumor necrosis factor alpha and gamma interferon), pathology, and antral-predominant infection were indistinguishable between IL-12-deficient and C57BL/6 mice. The increased permissiveness of the IL-12-deficient mouse for infection with
H. pylori
should facilitate whole-genome-based strategies to study genes associated with virulence and immune modulation.
A Constitutively Active Variant of the Quorum-Sensing Regulator LuxO Affects Protease Production and Biofilm Formation in
Vibrio cholerae
Authors: Vance Russell E. , Zhu Jun , Mekalanos John J.
Published: April 2003

Vibrio cholerae
normally inhabits aquatic habitats but can cause a severe diarrheal illness in humans. Its arsenal of virulence factors includes a secreted hemagglutinin (HA) protease. An HA protease-deficient mutant of
V. cholerae
was isolated and designated E7946
mpc.
E7946
mpc
was found to contain a point mutation in the
luxO
quorum-sensing regulator. In accordance with this finding, E7946
mpc
exhibits a defect in quorum sensing. The mutant
luxO
allele [
luxO
(Con)] produces a protein with a leucine-to-glutamine substitution at amino acid 104. Transfer of the
luxO
(Con) allele to an otherwise wild-type background was sufficient to eliminate HA protease expression; conversely, deletion of
luxO
(Con) from E7946
mpc
restored protease activity. We demonstrate that LuxO(Con) constitutively represses the transcription of
hapR
, an essential positive regulator of HA protease. Interestingly, strains harboring
luxO
(Con) form enhanced biofilms, and enhanced biofilm formation does not appear to be dependent on reduced HA protease expression. Taken together, the results confirm the role of LuxO as a central “switch” that coordinately regulates virulence-related phenotypes such as protease production and biofilm formation.