bluuebunny/crossref_metadata_2025 huggingface dataset:
Determining the Distribution of Hydraulic Conductivity in a Fractured Limestone Aquifer by Simultaneous Injection and Geophysical Logging
Authors: Morin Roger H. , Hess Alfred E. , Paillet Frederick L.
Published: March 2006

A field technique for assessing the vertical distribution of hydraulic conductivity in an aquifer was applied to a fractured carbonate formation in southeastern Nevada. The technique combines the simultaneous use of fluid injection and geophysical logging to measure in situ vertical distributions of fluid velocity and hydraulic head down the borehole; these data subsequently are analyzed to arrive at quantitative estimates of hydraulic conductivity across discrete intervals in the aquifer. The diagnostic capabilities of the analysis were found to be constrained by the accuracy and resolution of the logging tools as well as by the hydraulic characteristics of the formation. Despite these limitations, this injection/logging technique appears to be practical and effective under the conditions encountered in this application. The results of this analysis identified the contact margin between the Anchor and Dawn Members of the Monte Cristo Limestone as being the dominant transmissive unit. This section is extensively fractured, and quantitative estimates of its hydraulic conductivity are at least two orders of magnitude larger than that of the surrounding competent rock matrix. This transmissive zone also correlates strongly with an interval where the drilling rate was reported to be quite rapid. Aquifer transmissivity was computed from the profile of hydraulic conductivity and found to be 7.6 × 105 ft2/day, a value that is in close agreement with that determined from a conventional constant‐discharge test. Discrepancies between the two estimates may be scale‐dependent, a function of the different formation volumes investigated by each method. The friction factor, a parameter derived from the differential pressure and velocity logs, appears to be a good indicator of borehole rugosity across intervals where fluid velocity remains virtually unchanged, and vertical flow is turbulent.
Hydrogeology of Clay Till in a Prairie Region of Canada
Authors: Hendry M. J.
Published: March 2006

Hydrogeologic and isotopic investigations were conducted on thick profiles of clayey glacial till at three study areas in the Interior Plains Region of southern Alberta, Canada. The till consists of an upper weathered zone (9 to 18 m thick) and a lower nonweathered zone (10 to 30 m thick). The presence of tritiated ground water at depths of up to 5 m below the water table in the weathered zones shows that the upper part of the weathered zone is hydrogeologically active. Interpretation of hydraulic head and hydraulic conductivity data, indicates that vertical seepage velocities in the nonweathered till zones range from 2 to 6 m per 1000 years. Calculated lateral ground‐water velocities in the till zones are only about 9 m per 1000 years, and it is concluded that most of the ground water recharging the weathered till zone is returned to the atmosphere by evapotranspiration. Oxygen‐18 and deuterium analyses of ground‐water samples for the weathered till zones support this conclusion.
Simulation and Parameter Identification of Mass Transport in Ground Water
Authors: Häfner F. , Schwan M.
Published: March 2006

The transport of solute components in ground water takes place under the effects of convection, dispersion, and interactions between the stagnant water phase and the solid matrix. The inflow of solutes occurs in the form of local or diffuse sources. Among these, areal sources (precipitation) possess special importance in the recharging of ground water. Today, simulation of the process with multidimensional models is only possible with numerical methods. Since convective transport predominates in many cases, a one‐dimensional simulation scheme along the flow path is proposed for steady‐state ground‐water flow, which is suited to personal computers. This scheme represents the basis for the identification of migration parameters (inverse mass transport problem).
Synthetic Diether‐linked Cationic Lipids for Gene Delivery
Authors: Liu Dongliang , Qiao Weihong , Li Zongshi , Zhang Shubiao , Cheng Lvbo , Jin Kun
Published: March 2006

Quaternary ammonium lipids 2a–p, with diether linkages between hydrocarbon chains and their ammonium headgroups, were synthesized as potential vectors for cationic liposome‐mediated gene delivery. Varying the length of carbon chains and quaternary ammonium heads as well as different anionic complexes will enable the study of the structure–function relationships of these cationic lipids in terms of gene delivery properties.
Synthesis and Molecular Modelling of Novel Substituted‐4,5‐dihydro‐(1H)‐pyrazole Derivatives as Potent and Highly Selective Monoamine Oxidase‐A Inhibitors
Authors: Chimenti Franco , Bolasco Adriana , Manna Fedele , Secci Daniela , Chimenti Paola , Granese Arianna , Befani Olivia , Turini Paola , Alcaro Stefano , Ortuso Francesco
Published: March 2006

This report describes novel pyrazoline derivatives investigated for their ability to selectively inhibit the activity of the A and B isoforms of monoamine oxidase. These new synthetic compounds proved to be reversible, potent, and selective inhibitors of monoamine oxidase‐A rather than of monoamine oxidase‐B, and are promising candidates to further advance drug discovery efforts. The most active compounds show inhibitory activity on monoamine oxidase‐A in the 1.0 × 10−8–8.6 ×  10−9 mrange. Moreover, it should be pointed out that for some compounds a high IC50 ≥ 10−9 m value is associated with a high A‐selectivity (Selectivity Index monoamine oxidase‐B/monoamine oxidase‐A in the 10 000–12 500 range). Further insight to understand enzyme‐inhibitor molecular interaction was obtained by docking experiments with the monoamine oxidase‐A and monoamine oxidase‐B isoforms.
Valproic Acid Promotes Neurite Outgrowth in PC12 Cells independent from Regulation of the Survival of Motoneuron Protein
Authors: van Bergeijk Jeroen , Haastert Kirsten , Grothe Claudia , Claus Peter
Published: March 2006

Spinal muscular atrophy (SMA) is a neurodegenerative disorder of motoneurons. The disease is caused by deletions or mutations of the survival of motoneuron gene 1 (SMN1). The amount of protein expressed from the second gene, SMN2, correlated with the severity of the clinical phenotype. The histone deacetylase inhibitor valproic acid (VPA) has been shown to increase the total cellular amount of functional SMN protein and is therefore considered as a drug candidate for treatment of SMA. In this study, we analyzed the effects of VPA in PC12 cells, a model system for neuronal differentiation, with regard to neurite outgrowth and SMN expression. VPA promoted neurite outgrowth in PC12 cells. However, this effect did not correlate with upregulation of SMN protein levels, suggesting a SMN‐independent mechanism for VPA regulation of neurite outgrowth.
Perspectives on Health Among Adult Users of Illicit Stimulant Drugs in Rural Ohio
Authors: Siegal Harvey A. , Draus Paul J. , Carlson Robert G. , Falck Russel S. , Wang Jichuan
Published: March 2006

ABSTRACT: 
Context: Although the nonmedical use of stimulant drugs such as cocaine and methamphetamine is increasingly common in many rural areas of the United States, little is known about the health beliefs of people who use these drugs. Purpose: This research describes illicit stimulant drug users’ views on health and health‐related concepts that may affect their utilization of health care services. Methods: A respondent‐driven sampling plan was used to recruit 249 not‐in‐treatment, nonmedical stimulant drug users who were residing in 3 rural counties in west central Ohio. A structured questionnaire administered by trained interviewers was used to collect information on a range of topics, including current drug use, self‐reported health status, perceived need for substance abuse treatment, and beliefs about health and health services. Findings: Participants reported using a wide variety of drugs nonmedically, some by injection. Alcohol and marijuana were the most commonly used drugs in the 30 days prior to the interview. Powder cocaine was used by 72.3% of the sample, crack by 68.3%, and methamphetamine by 29.7%. Fair or poor health status was reported by 41.3% of the participants. Only 20.9% of the sample felt they needed drug abuse treatment. Less than one third of the sample reported that they would feel comfortable talking to a physician about their drug use, and 65.1% said they preferred taking care of their problems without getting professional help. Conclusions: Stimulant drug users in rural Ohio are involved with a range of substances and hold health beliefs that may impede health services utilization.
Gating of the expressed T‐type Cav3.1 calcium channels is modulated by Ca2+
Authors: Lacinová L. , Kurejová M. , Klugbauer N. , Hofmann F.
Published: March 2006

Aim:  We have investigated the influence of Ca2+ ions on the basic biophysical properties of T‐type calcium channels.

Methods:  The Cav3.1 calcium channel was transiently expressed in HEK 293 cells. Current was measured using the whole cell patch clamp technique. Ca2+ or Na+ ions were used as charge carriers. The intracellular Ca2+ was either decreased by the addition of 10 mm ethyleneglycoltetraacetic acid (EGTA) or increased by the addition of 200 μm Ca2+ into the non‐buffered intracellular solution. Various combinations of extra‐ and intracellular solutions yielded high, intermediate or low intracellular Ca2+ levels.

Results:  The amplitude of the calcium current was independent of intracellular Ca2+ concentrations. High levels of intracellular Ca2+ accelerated significantly both the inactivation and the activation time constants of the current. The replacement of extracellular Ca2+ by Na+ as charge carrier did not affect the absolute value of the activation and inactivation time constants, but significantly enhanced the slope factor of the voltage dependence of the inactivation time constant. Slope factors of voltage dependencies of channel activation and inactivation were significantly enhanced. The recovery from inactivation was faster when Ca2+ was a charge carrier. The number of available channels saturated for membrane voltages more negative than −100 mV for the Ca2+ current, but did not reach steady state even at −150 mV for the Na+ current.

Conclusions:  Ca2+ ions facilitate transitions of Cav3.1 channel from open into closed and inactivated states as well as backwards transition from inactivated into closed state, possibly by interacting with its voltage sensor.
Detecting the effects of introduced species: a case study of competition between Apis and Bombus
Authors: Thomson Diane M.
Published: March 2006

Developing tools for rapid assessment of introduced species impacts is one of the most important challenges in invasion ecology. Most assessments of impact rely on correlational data or other indirect measures. Yet few studies have evaluated invasion effects using multiple, simultaneously applied monitoring and experimental approaches, in order to compare easily obtained metrics with more difficult but direct measures of reproductive success or population dynamics. In this study, I use data from an experimental test of introduced honey bee (Apis mellifera) impacts on native bumble bees (Bombus spp.) to address two major questions: 1) how well did observational data on niche overlap and spatial correlations between Apis and Bombus predict the results of experimental tests of competitive effects? and 2) how well did effects of the experimental Apis manipulations on Bombus foragers, which are easy to observe, predict changes in reproductive success of colonies, which are difficult to measure? Niche overlap between Apis and Bombus varied substantially, but increased to levels as high as 80–90% during periods of resource scarcity. Correlations between numbers of Apis foragers and numbers of Bombus foragers were also highly variable, but I detected a significant negative relationship in only one of the seven months observed. In contrast, the experimental results showed that mean numbers of Bombus foragers observed on a given transect increased significantly with greater distance from introduced Apis colonies. Of these three measures (niche overlap, correlations in abundances, and effects of experimental introductions), only the experimental data on forager abundances accurately estimated competitive effects on colony reproductive success previously reported for the same experiment, and the correlational data in particular completely failed to predict the effects observed in the experimental study. This work suggests that great caution is warranted in making assessments of invasion impact on the basis of spatial or temporal correlations between invasive and native species. Thus, investing in even small and limited experimental studies may be more valuable than extensive observational work in quantifying invasion impacts.
Probing Cellular Chemistry in Biological Systems with Microelectrodes
Authors: Wightman R. Mark
Published: March 2006

Over the past 20 years, the technological impediments to fabricating electrodes of micrometer dimensions have been largely overcome. These small electrodes can be readily applied to probe chemical events at the surface of tissues or individual biological cells; they can even be used to monitor concentration changes within intact animals. These measurements can be made on rapid time scales and with minimal perturbation of the system under study. Several recent applications have provided important insights into chemical processes at cells and in tissues. Examples include molecular flux measurements at the surface of single cells and through skin—which can offer insights into oxidative stress, exocytosis, and drug delivery—and real-time brain neurotransmitter monitoring in living rats, which reveals correlations between behavior and molecular events in the brain. Such findings can promote interdisciplinary collaborations and may lead to a broader understanding of the chemical aspects of biology.
Leukotoxin Confers Beta-Hemolytic Activity to
Actinobacillus actinomycetemcomitans
Authors: Balashova Nataliya V. , Crosby Juan A. , Al Ghofaily Lourdes , Kachlany Scott C.
Published: March 2006

Actinobacillus actinomycetemcomitans
is the etiologic agent of localized aggressive periodontitis, a rapidly progressing oral disease that occurs in adolescents.
A. actinomycetemcomitans
can also cause systemic disease, including infective endocarditis. In early work on
A. actinomycetemcomitans
workers concluded that this bacterium is not beta-hemolytic. More recent reports have suggested that
A. actinomycetemcomitans
does have the potential to be beta-hemolytic. While growing
A. actinomycetemcomitans
on several types of growth media, we noticed a beta-hemolytic reaction on media from one manufacturer. Beta-hemolysis occurred on Columbia agar from Accumedia with either sheep or horse blood, but not on similar media from other manufacturers. A surprising result was that mutants of
A. actinomycetemcomitans
defective for production of leukotoxin, a toxin that is reportedly highly specific for only human and primate white blood cells, are not beta-hemolytic. Purified leukotoxin was able to lyse sheep and human erythrocytes in vitro. This work showed that in contrast to the accepted view,
A. actinomycetemcomitans
leukotoxin can indeed destroy erythrocytes and that the production of this toxin results in beta-hemolytic colonies on solid medium. In light of these results, the diagnostic criteria for clinical identification of
A. actinomycetemcomitans
and potentially related bacteria should be reevaluated. Furthermore, in studies on
A. actinomycetemcomitans
leukotoxin workers should now consider this toxin's ability to destroy red blood cells.
Analysis of Involvement of the RecF Pathway in
p44
Recombination in
Anaplasma phagocytophilum
and in
Escherichia coli
by Using a Plasmid Carrying the
p44
Expression and
p44
Donor Loci
Authors: Lin Quan , Zhang Chunbin , Rikihisa Yasuko
Published: March 2006

Anaplasma phagocytophilum
, the etiologic agent of human granulocytic anaplasmosis, has a large paralog cluster (approximate 90 members) that encodes the 44-kDa major outer membrane proteins (P44s). Gene conversion at a single
p44
expression locus leads to P44 antigenic variation. Homologs of genes for the RecA-dependent RecF pathway, but not the RecBCD or RecE pathways, of recombination were detected in the
A. phagocytophilum
genome. In the present study, we examined whether the RecF pathway is involved in
p44
gene conversion. The recombination intermediate structure between a donor
p44
and the
p44
expression locus of
A. phagocytophilum
was detected in an HL-60 cell culture by Southern blot analysis followed by sequencing the band and in blood samples from infected SCID mice by PCR, followed by sequencing. The sequences were consistent with the RecF pathway recombination: a half-crossover structure, consisting of the donor
p44
locus connected to the 3′ conserved region of the recipient
p44
in the
p44
expression locus in direct orientation. To determine whether the
p44
recombination intermediate structure can be generated in a RecF-active
Escherichia coli
strain, we constructed a double-origin plasmid carrying the
p44
expression locus and a donor
p44
locus and introduced the plasmid into various
E. coli
strains. The recombination intermediate was recovered in an
E. coli
strain with active RecF recombination pathway but not in strains with deficient RecF pathway. Our results support the view that the
p44
gene conversion in
A. phagocytophilum
occurs through the RecF pathway.
Intranasal Vaccination with a Defined AttenuatedFrancisella novicidaStrain Induces Gamma Interferon-Dependent Antibody-Mediated Protection against Tularemia
Authors: Pammit Michael A. , Raulie Erin K. , Lauriano Crystal M. , Klose Karl E. , Arulanandam Bernard P.
Published: March 2006

Francisella tularensisis an intracellular gram-negative bacterium that is the causative agent of tularemia and a potential bioweapon. We have characterized the efficacy of a definedF. novicidamutant (ΔiglC) as a live attenuated vaccine against subsequent intranasal challenge with the wild-type organism. Animals primed with theF. novicida ΔiglC(KKF24) mutant induced robust splenic gamma interferon (IFN-γ) and interleukin-12 (IL-12) recall responses with negligible IL-4 production as well as the production of antigen-specific serum immunoglobulin G1 (IgG1) and IgG2a antibodies. BALB/c mice vaccinated intranasally (i.n.) with KKF24 and subsequently challenged with wild-typeF. novicida(100 and 1,000 50% lethal doses) were highly protected (83% and 50% survival, respectively) from the lethal challenges. The protection conferred by KKF24 vaccination was shown to be highly dependent on endogenous IFN-γ production and also was mediated by antibodies that could be adoptively transferred to naive B-cell-deficient mice by inoculation of immune sera. Collectively, the results demonstrate that i.n. vaccination with KKF24 induces a vigorous Th1-type cytokine and antibody response that is protective against subsequent i.n. challenge with the wild-type strain. This is the first report of a defined live attenuated strain providing protection against the inhalation ofF. novicida.
An Increase in Antimycobacterial Th1-Cell Responses by Prime-Boost Protocols of Immunization Does Not Enhance Protection against Tuberculosis
Authors: Majlessi Laleh , Simsova Marcela , Jarvis Zdenka , Brodin Priscille , Rojas Marie-Jésus , Bauche Cécile , Nouzé Clémence , Ladant Daniel , Cole Stewart T. , Sebo Peter , Leclerc Claude
Published: March 2006

Bordetella pertussisadenylate cyclase (CyaA) toxoid is a powerful nonreplicative immunization vector targeting dendritic cells, which has already been used successfully in prophylactic and therapeutic vaccination in various preclinical animal models. Here, we investigated the potential of CyaA, harboring strong mycobacterial immunogens, i.e., the immunodominant regions of antigen 85A or the complete sequence of the 6-kDa early secreted antigenic target (ESAT-6) protein, to induce antimycobacterial immunity. By generating T-cell hybridomas or by using T cells from mice infected with mycobacteria, we first demonstrated that the in vitro delivery of 85A or ESAT-6 to antigen-presenting cells by CyaA leads to processing and presentation, by major histocompatibility complex class II molecules, of the same epitopes as those displayed upon mycobacterial infection. Importantly, compared to the recombinant protein alone, the presentation of ESAT-6 in vitro was 100 times more efficient upon its delivery to antigen-presenting cells in fusion to CyaA. Immunization with CyaA-85A or CyaA-ESAT-6 in the absence of any adjuvant induced strong antigen-specific lymphoproliferative, interleukin-2 (IL-2) and gamma interferon (IFN-γ) cytokine responses, in the absence of any IL-4 or IL-5 production. When used as boosters after priming with a BCG expressing ESAT-6, the CyaA-85A and CyaA-ESAT-6 proteins were able to strikingly increase the sensitivity and intensity of proliferative and Th1-polarized responses and notably the frequency of antigen-specific IFN-γ-producing CD4+T cells. However, immunization with these CyaA constructs as subunit vaccines alone or as boosters did not allow induction or improvement of protection againstMycobacterium tuberculosisinfection. These results question the broadly admitted correlation between the frequency of IFN-γ-producing CD4+T cells and the level of protection against tuberculosis.
Immunization with
Staphylococcus aureus
Clumping Factor B, a Major Determinant in Nasal Carriage, Reduces Nasal Colonization in a Murine Model
Authors: Schaffer Adam C. , Solinga Robert M. , Cocchiaro Jordan , Portoles Marta , Kiser Kevin B. , Risley Allison , Randall Suzanne M. , Valtulina Viviana , Speziale Pietro , Walsh Evelyn , Foster Timothy , Lee Jean C.
Published: March 2006

Staphylococcus aureus
is responsible for a wide range of infections, including soft tissue infections and potentially fatal bacteremias. The primary niche for
S. aureus
in humans is the nares, and nasal carriage is a documented risk factor for staphylococcal infection. Previous studies with rodent models of nasal colonization have implicated capsule and teichoic acid as staphylococcal surface factors that promote colonization. In this study, a mouse model of nasal colonization was utilized to demonstrate that
S. aureus
mutants that lack clumping factor A, collagen binding protein, fibronectin binding proteins A and B, polysaccharide intercellular adhesin, or the accessory gene regulator colonized as well as wild-type strains colonized. In contrast, mutants deficient in sortase A or clumping factor B (ClfB) showed reduced nasal colonization. Mice immunized intranasally with killed
S. aureus
cells showed reduced nasal colonization compared with control animals. Likewise, mice that were immunized systemically or intranasally with a recombinant vaccine composed of domain A of ClfB exhibited lower levels of colonization than control animals exhibited. A ClfB monoclonal antibody (MAb) inhibited
S. aureus
binding to mouse cytokeratin 10. Passive immunization of mice with this MAb resulted in reduced nasal colonization compared with the colonization observed after immunization with an isotype-matched control antibody. The mouse immunization studies demonstrate that ClfB is an attractive component for inclusion in a vaccine to reduce
S. aureus
nasal colonization in humans, which in turn may diminish the risk of staphylococcal infection. As targets for vaccine development and antimicrobial intervention are assessed, rodent nasal colonization models may be invaluable.
Pore-Forming and Enzymatic Activities ofBordetella pertussisAdenylate Cyclase Toxin Synergize in Promoting Lysis of Monocytes
Authors: Basler Marek , Masin Jiri , Osicka Radim , Sebo Peter
Published: March 2006

Bordetellaadenylate cyclase (AC) toxin-hemolysin (CyaA) targets myeloid phagocytes expressing the αMβ2integrin (CD11b/CD18) and delivers into their cytosol an AC enzyme that converts ATP into cyclic AMP (cAMP). In parallel, CyaA acts as a hemolysin, forming small membrane pores. Using specific mutations, we dissected the contributions of the two activities to cytolytic potency of CyaA on J774A.1 murine monocytes. The capacity of AC to penetrate cells and deplete cytosolic ATP was essential for promoting lysis and the enzymatically inactive but fully hemolytic CyaA-AC−toxoid exhibited a 15-fold-lower cytolytic capacity on J774A.1 cells than intact CyaA. Moreover, a two- or fourfold drop of specific hemolytic activity of the CyaA-E570Q and CyaA-E581P mutants was overpowered by an intact capacity to dissipate cytosolic ATP into cAMP, allowing the less hemolytic proteins to promote lysis of J774A.1 cells as efficiently as intact CyaA. However, an increased hemolytic activity, due to lysine substitutions of glutamates 509, 516, and 581 in the pore-forming domain, conferred on AC−toxoids a correspondingly enhanced cytolytic potency. Moreover, a threefold increase in hemolytic activity could override a fourfold drop in capacity to convert cellular ATP to cAMP, conferring on the CyaA-E581K construct an overall twofold increased cytolytic potency. Hence, although appearing auxiliary in cytolytic action of the toxin on nucleated cells, the pore-forming activity can synergize with ATP-depleting activity of the cell-invasive AC enzyme and complement its action toward maximal cytotoxicity.
A Novel
Staphylococcus aureus
Vaccine: Iron Surface Determinant B Induces Rapid Antibody Responses in Rhesus Macaques and Specific Increased Survival in a Murine
S. aureus
Sepsis Model
Authors: Kuklin Nelly A. , Clark Desmond J. , Secore Susan , Cook James , Cope Leslie D. , McNeely Tessie , Noble Liliane , Brown Martha J. , Zorman Julie K. , Wang Xin Min , Pancari Gregory , Fan Hongxia , Isett Kevin , Burgess Bruce , Bryan Janine , Brownlow Michelle , George Hugh , Meinz Maria , Liddell Mary E. , Kelly Rosemarie , Schultz Loren , Montgomery Donna , Onishi Janet , Losada Maria , Martin Melissa , Ebert Timothy , Tan Charles Y. , Schofield Timothy L. , Nagy Eszter , Meineke Andreas , Joyce Joseph G. , Kurtz Myra B. , Caulfield Michael J. , Jansen Kathrin U. , McClements William , Anderson Annaliesa S.
Published: March 2006

Staphylococcus aureus
is a major cause of nosocomial infections worldwide, and the rate of resistance to clinically relevant antibiotics, such as methicillin, is increasing; furthermore, there has been an increase in the number of methicillin-resistant
S. aureus
community-acquired infections. Effective treatment and prevention strategies are urgently needed. We investigated the potential of the
S. aureus
surface protein iron surface determinant B (IsdB) as a prophylactic vaccine against
S. aureus
infection. IsdB is an iron-sequestering protein that is conserved in diverse
S. aureus
clinical isolates, both methicillin resistant and methicillin sensitive, and it is expressed on the surface of all isolates tested. The vaccine was highly immunogenic in mice when it was formulated with amorphous aluminum hydroxyphosphate sulfate adjuvant, and the resulting antibody responses were associated with reproducible and significant protection in animal models of infection. The specificity of the protective immune responses in mice was demonstrated by using an
S. aureus
strain deficient for IsdB and HarA, a protein with a high level of identity to IsdB. We also demonstrated that IsdB is highly immunogenic in rhesus macaques, inducing a more-than-fivefold increase in antibody titers after a single immunization. Based on the data presented here, IsdB has excellent prospects for use as a vaccine against
S. aureus
disease in humans.
Efficient Translocation of EspC into Epithelial Cells Depends on Enteropathogenic
Escherichia coli
and Host Cell Contact
Authors: Vidal Jorge E. , Navarro-García Fernando
Published: March 2006

EspC is an autotransporter protein secreted by enteropathogenic
Escherichia coli
(EPEC). The pathogenic role of EspC in EPEC infection is unknown. We have shown that the purified EspC produces enterotoxicity and cytotoxicity; for the latter effect, EspC must be internalized. However, the internalization mechanism is unknown. Here we show that azithromycin (an inhibitor of pinocytosis), but not drugs affecting caveole-, clathrin-, or receptor-mediated endocytosis, inhibited purified EspC internalization and cytoskeletal disruption, suggesting that purified EspC is internalized by pinocytosis. Furthermore, unlike in cholera toxin, we were unable to detect a receptor on epithelial cells by pretreatment at 4°C. Upon EspC entry, it is delivered directly into the cell cytosol, as shown by the fact that drugs that inhibit intracellular trafficking had no effect on cytoskeletal disruption. All these data suggest that purified EspC internalization is not a physiological internalization mechanism; hence, we explored EspC internalization during the infection of epithelial cells by EPEC. Like other EPEC virulence factors, EspC secretion is stimulated by EPEC when it is grown in cell culture medium and enhanced by the presence of epithelial cells. Physiologically secreted EspC was efficiently internalized during EPEC and host cell interaction. Additionally, the lack of EspC internalization caused by using an isogenic mutant prevented the cytopathic effect caused by EPEC. These data suggest that EPEC uses an efficient mechanism to internalize milieu-secreted EspC into epithelial cells; once inside the cells, EspC is able to induce the cytopathic effect caused by EPEC.
Characterization ofBacteroides fragilisHemolysins and Regulation and Synergistic Interactions of HlyA and HlyB
Authors: Robertson Kirstin P. , Smith C. Jeffrey , Gough Andrea M. , Rocha Edson R.
Published: March 2006

This study describes the presence of 10 hemolysin orthologs in the genome of the opportunistic human anaerobic pathogenBacteroides fragilis, which is currently classified as a nonhemolytic bacterium. The hemolysins were designated HlyA through HlyI plus HlyIII. All cloned hemolysin genes were able to confer hemolytic activity to a nonhemolyticEscherichia colistrain on blood agar plates. Interestingly, HlyH was found to be present in the genome of theB. fragilisNCTC9343 strain but absent in strains 638R, YCH46, andBacteroides thetaiotaomicronVPI-5482. The hemolysins HlyA, HlyB, and HlyIII were selected for further characterization. HlyA, HlyB, and HlyIII were cytolytic to erythrocytes on liquid hemolytic assay. WhenhlyAandhlyBwere expressed together in a nonhemolyticE. colistrain, the strain showed enhanced hemolytic activity on blood agar plates. Further analysis revealed that HlyA and HlyB have synergistic hemolytic activity as detected by the liquid hemolytic assay. In addition, the two-component hemolysins HlyA and HlyB form a protein-protein complex in vivo as determined by bacterial two-hybrid system assay. ThehlyBandhlyAgenes are organized in an operon that is coordinately regulated by iron and oxygen. Northern blot hybridization analysis revealed thathlyBAwere expressed as a bicistronic mRNA induced approximately 2.5-fold under low-iron conditions and repressed in iron-rich medium. The normal iron-regulated expression ofhlyBAmRNA was lost in thefurAmutant strain. In contrast, thehlyAgene was also expressed as a single mRNA in iron-rich medium, but its expression was reduced approximately threefold under low-iron conditions in a Fur-independent manner. This suggests thathlyAalone is regulated by an unidentified iron-dependent regulator. Moreover, the expression levels ofhlyBAandhlyAwere reduced about threefold following oxygen exposure and treatment with hydrogen peroxide. Taken together, these results suggest that iron and oxidative stress have an effect on the control ofhlyBAandhlyAtranscriptional levels. AhlyBAmutant was constructed, and its hemolytic activity was greatly diminished compared to those of thehlyIIImutant and parent strains. In addition, thehlyBAmutant had a significant modification in colony morphology and growth deficiency compared to the parent strain. The implications of these findings for the pathophysiology ofB. fragilisin extraintestinal infections and competition in ecological systems for this organism are discussed.
ShiA Abrogates the Innate T-Cell Response toShigella flexneriInfection
Authors: Ingersoll Molly A. , Zychlinsky Arturo
Published: March 2006

Shigellaspp. are the causative agent of bacillary dysentery. Infection results in acute colonic injury due to the host inflammatory response. The mediators of the damage, infiltrating polymorphonuclear leukocytes (PMN), also resolve the infection.Shigella flexneri's virulence effectors are encoded on its large virulence plasmid and on pathogenicity islands in the chromosome. The SHI-2 pathogenicity island encodes the virulence factor ShiA, which down-regulatesShigella-induced inflammation. In the rabbit ileal loop model, infection with ashiAnull strain (ΔshiA) induces a more severe inflammation than wild-type infection. Conversely, aShigellastrain that overexpresses ShiA (ShiA+) is less inflammatory than the wild-type strain. To determine the host responses modulated by ShiA, we performed infection studies using the mouse lung model, which recapitulates the phenotypes observed in the rabbit ileal loop model. Significantly, ShiA+strain-infected mice cleared the bacteria and survived infection, while wild-type- and ΔshiAstrain-infected mice could not clear the bacteria and ultimately died. Surprisingly, microarray analysis of infected lungs revealed the regulation of genes involved in innate T-cell responses to infection. Immunohistochemistry showed that wild-type- and ΔshiAstrain-infected animals have greater numbers of PMN and T cells in their lungs over the course of infection than ShiA+strain-infected animals. These results suggest that the T-cell innate response is suppressed by ShiA inShigellainfections.
Essential Role of the Type III Secretion System Effector NleB in Colonization of Mice by
Citrobacter rodentium
Authors: Kelly Michelle , Hart Emily , Mundy Rosanna , Marchès Olivier , Wiles Siouxsie , Badea Luminita , Luck Shelley , Tauschek Marija , Frankel Gad , Robins-Browne Roy M. , Hartland Elizabeth L.
Published: March 2006

Attaching and effacing (A/E) pathogens are a significant cause of gastrointestinal illness in humans and animals. All A/E pathogens carry a large pathogenicity island, termed the locus for enterocyte effacement (LEE), which encodes a type III secretion system that translocates several effector proteins into host cells. To identify novel virulence determinants in A/E pathogens, we performed a signature-tagged mutagenesis screen in C57BL/6 mice by using the mouse A/E pathogen
Citrobacter rodentium
. Five hundred seventy-six derivatives of
C. rodentium
were tested in pools of 12 mutants. One attenuated mutant carried a transposon insertion in
nleB
, which encodes a putative effector of the LEE-encoded type III secretion system (T3SS).
nleB
is present in a genomic pathogenicity island that also encodes another putative effector, NleE, immediately downstream. Using translational fusions with β-lactamase (TEM-1), we showed that both NleB and NleE were translocated into host cells by the LEE-encoded T3SS of enteropathogenic
Escherichia coli
. In addition, deletion of the gene encoding NleB in
C. rodentium
resulted in reduced colonization of mice in single infections and reduced colonic hyperplasia. In contrast, the deletion of other non-LEE-encoded effector genes in
C. rodentium
,
nleC
,
nleD
, or
nleE
, had no effect on host colonization or disease. These results suggest that
nleB
encodes an important virulence determinant of A/E pathogens.
Topical H2 Antagonist Prevents Periodontitis in a Rabbit Model
Authors: Hasturk H. , Kantarci A. , Ebrahimi N. , Andry C. , Holick M. , Jones V. L. , Van Dyke T. E.
Published: March 2006

Cimetidine is a powerful H2 receptor antagonist that eliminates histamine's effects on chemotaxis, phagocytosis, and superoxide anion production by phagocytes. The purpose of this study was to analyze the clinical and histopathological changes associated with experimental periodontitis in rabbits in response to topically applied cimetidine. Experimental periodontitis was induced in 21 New Zealand White rabbits usingPorphyromonas gingivalis(109CFU) topically applied three times a week for a 6-week period to previously ligatured teeth. Topical application of cimetidine in a liposome carrier for the prevention of periodontitis was evaluated in four groups of four animals each: 1, 10, and 100 mg/ml and no treatment (positive control). In addition, there was a vehicle group (n= 3) that received liposome preparation (carrier) only, and two animals with ligature application alone served as negative controls. Periodontal disease was quantified by direct visualization and radiographical evaluation of bone loss on defleshed skulls and by histological analyses of sections stained with hematoxylin-eosin and tartrate-resistant acid phosphatase. In the no-treatment (positive control) and liposome (vehicle) groups, direct visualization and radiological measurements revealed statistically significant bone loss compared to the negative control. Application of cimetidine at all concentrations tested inhibited inflammation and bone loss by >90%. Histological findings revealed that ligated sites of the positive control and vehicle groups showed significant reduction in bone level (P< 0.05) compared to the three cimetidine groups, with a marked decrease in inflammation. The findings of this study provide morphological and histological evidence that topically active cimetidine is a potent inhibitor ofP. gingivalis-elicited periodontal inflammation, arresting and/or preventing tissue destruction and influencing cell populations present in the inflammatory cell infiltrate.
Spherules Derived from
Coccidioides posadasii
Promote Human Dendritic Cell Maturation and Activation
Authors: Dionne Sara O. , Podany Abigail B. , Ruiz Yvette W. , Ampel Neil M. , Galgiani John N. , Lake Douglas F.
Published: March 2006

Previous studies have shown that dendritic cells (DC) pulsed with T27K, an antigenic preparation derived from spherules (of
Coccidioides posadasii
), activate peripheral blood mononuclear cells (PBMC) from nonimmune subjects as well as from patients with disseminated coccidioidomycosis. In this study, we have assessed the interaction between human DC and
C. posadasii
spherules in order to better understand the initial response between
Coccidioides
and the human host. Whole autoclaved spherules induced lymphocyte transformation in PBMC obtained from immune but not from nonimmune donors. Immature DC (iDC) bound fluorescein isothiocyanate-labeled spherules in a time- and temperature-dependent manner. This binding was blocked by the addition of mannan, suggesting mannose receptor involvement in the DC-
Coccidioides
interaction. Binding was subsequently associated with ingestion and intracellular processing of spherules. Coculturing of spherules with iDC was associated with the development of mature DC that were morphologically, phenotypically, and functionally similar to those induced by tumor necrosis factor alpha and prostaglandin E
2
. Finally, spherules incubated with iDC induced activation of PBMC from nonimmune donors. These data indicate that human DC are capable of binding, internalizing, and presenting antigens from
Coccidioides
spherules and suggest that DC may play a critical early role in the formation of a cellular immune response in human coccidioidomycosis.
RrgA and RrgB Are Components of a Multisubunit Pilus Encoded by theStreptococcus pneumoniae rlrAPathogenicity Islet
Authors: LeMieux Julianna , Hava David L. , Basset Alan , Camilli Andrew
Published: March 2006

TherlrApathogenicity islet inStreptococcus pneumoniaeTIGR4 encodes three surface proteins, RrgA, RrgB, and RrgC, and three sortase enzymes. Using transmission electron microscopy, cell fractionation, cell wall sorting signal domain swapping, and Western blotting, we show that RrgA and RrgB are incorporated into a multisubunit pilus inS. pneumoniae.
Posttranslational Regulation of
Mycobacterium tuberculosis
Extracytoplasmic-Function Sigma Factor σ
L
and Roles in Virulence and in Global Regulation of Gene Expression
Authors: Dainese Elisa , Rodrigue Sébastien , Delogu Giovanni , Provvedi Roberta , Laflamme Liette , Brzezinski Ryszard , Fadda Giovanni , Smith Issar , Gaudreau Luc , Palù Giorgio , Manganelli Riccardo
Published: March 2006

In this report, we demonstrate that SigL is posttranslationally regulated by a specific anti-sigma factor, RslA, and contributes to the expression of at least 28 genes. Several of these genes could mediate important cell envelope-related processes. Importantly, a
sigL
-
rslA
mutant strain was significantly attenuated in a mouse model of infection.
The
Treponema denticola
Surface Protease Dentilisin Degrades Interleukin-1β (IL-1β), IL-6, and Tumor Necrosis Factor Alpha
Authors: Miyamoto Meguru , Ishihara Kazuyuki , Okuda Katsuji
Published: March 2006

Dentilisin is a major surface protease and virulence factor of the bacterium
Treponema denticola
. In this study, we found that
T. denticola
reduced inflammatory cytokines, including interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha, in peripheral blood mononuclear cells through degradation by dentilisin.
Distinct Kinetics of Effector CD8+Cytotoxic T Cells after Infection withTrypanosoma cruziin Naïve or Vaccinated Mice
Authors: Tzelepis Fanny , de Alencar Bruna C. G. , Penido Marcus L. O. , Gazzinelli Ricardo T. , Persechini Pedro M. , Rodrigues Mauricio M.
Published: March 2006

The kinetics of effector CD8+-T-cell responses to specificTrypanosoma cruziepitopes was investigated after challenge. Our results suggest that the delayed kinetics differs from that observed in other microbial infections and facilitates the establishment of the disease in naïve mice. In contrast, in vaccinated mice, the swift CD8+-T-cell response helps host survival after challenge.
SYSTEM ORIENTED NEURAL NETWORKS — PROBLEM FORMULATION, METHODOLOGY AND APPLICATION
Authors: LI KANG , PENG JIAN-XUN
Published: March 2006

A novel methodology is proposed for the development of neural network models for complex engineering systems exhibiting nonlinearity. This method performs neural network modeling by first establishing some fundamental nonlinear functions from a priori engineering knowledge, which are then constructed and coded into appropriate chromosome representations. Given a suitable fitness function, using evolutionary approaches such as genetic algorithms, a population of chromosomes evolves for a certain number of generations to finally produce a neural network model best fitting the system data. The objective is to improve the transparency of the neural networks, i.e. to produce physically meaningful "white box" neural network model with better generalization performance. In this paper, the problem formulation, the neural network configuration, and the associated optimization software are discussed in detail. This methodology is then applied to a practical real-world system to illustrate its effectiveness.
COMPUTATIONAL METHOD FOR PROTEIN FUNCTION PREDICTION BY CONSTRUCTING PROTEIN INTERACTION NETWORK DICTIONARY
Authors: JIN HEE-JEONG , CHO HWAN-GUE
Published: March 2006

In the post-genomic era, predicting protein function is a challenging problem. It is difficult and burdensome work to unravel the functions of a protein by wet experiments only. In this paper, we propose a novel method to predict protein functions by building a "Protein Interaction Network Dictionary (PIND)". This method deduces the protein functions by searching the most similar "words"(an anagram of functions in neighbor proteins on a protein–protein interaction graph) using global alignments. An evaluation of sensitivity and specificity shows that this PIND approach outperforms previous approaches such as Majority Rule and Chi-Square measure, and that it competes with the recently introduced Random Markov Model approach.
TUMOR CLASSIFICATION BASED ON INDEPENDENT COMPONENT ANALYSIS
Authors: ZHENG CHUN-HOU , CHEN YAN , LI XIU-XIA , LI YI-XUE , ZHU YUN-PING
Published: March 2006

This paper proposes a new method for tumor classification using gene expression data, which mainly contains three steps. Firstly, the original DNA microarray gene expression data are selected using t-statistics. Secondly, the selected genes are modeled by Independent Component Analysis (ICA). Finally, Support Vector Machine (SVM) is used to classify the modeling data. To show the validity of the proposed method, we apply it to classify two DNA microarray data sets involving various human normal and tumor tissue samples. The experimental results show that the method is efficient and feasible.
GENERICITY IN EVOLUTIONARY COMPUTATION SOFTWARE TOOLS: PRINCIPLES AND CASE-STUDY
Authors: GAGNÉ CHRISTIAN , PARIZEAU MARC
Published: March 2006

This paper deals with the need for generic software development tools in evolutionary computations (EC). These tools will be essential for the next generation of evolutionary algorithms where application designers and researchers will need to mix different combinations of traditional EC (e.g. genetic algorithms, genetic programming, evolutionary strategies, etc.), or to create new variations of these EC, in order to solve complex real world problems. Six basic principles are proposed to guide the development of such tools. These principles are then used to evaluate six freely available, widely used EC software tools. Finally, the design of Open BEAGLE, the framework developed by the authors, is presented in more detail.
MODEL CHECKING FOR MULTIAGENT SYSTEMS: THE MABLE LANGUAGE AND ITS APPLICATIONS
Authors: WOOLDRIDGE MICHAEL , HUGET MARC-PHILIPPE , FISHER MICHAEL , PARSONS SIMON
Published: March 2006

We present MABLE, a fully implemented programming language for multiagent systems, which is intended to support the automatic verification of such systems via model checking. In addition to the conventional constructs of imperative programming languages, MABLE provides a number of agent-oriented development features. First, agents in MABLE are endowed with a BDI-like mental state: they have data structures corresponding to beliefs, desires, and intentions, and these mental states may be arbitrarily nested. Second, agents in MABLE communicate via ACL-like performatives: however, neither the performatives nor their semantics are hardwired into the language. It is possible to define the performatives and the semantics of these performatives independently of the system in which they are used. Using this feature, a developer can explore the design space of ACL performatives and semantics without changing the target system. Finally, MABLE supports automatic verification via model checking. Claims about the behaviour of a MABLE system can be expressed in a linear-time BDI-like logic, and the truth, or otherwise, of these claims can be automatically determined. Following a description of the MABLE language and the language of MABLE claims, we present two case studies to illustrate the language and its use in the verification of multiagent systems. We then describe the key ideas underpinning the current implementation of MABLE. Finally, we survey related work, and discuss some avenues for future research.
MODULAR STRUCTURES AND ROBUSTNESS OF PROTEIN NETWORKS
Authors: NG KA-LOK , HUANG CHIEN-HUNG , LEE PO-HAN , FANG JYWE-FEI , TSAI JEFFREY J. P.
Published: March 2006

The topological structures and robustness of the protein-protein interaction networks (PINs) is studied via random graph theory. Several global topological parameters are used to characterize PINs for seven species. Good evidence by correlation analysis support the fact that the seven PINs are well approximated by scale-free networks. It is also found that two of the species' PINs are well described by the hierarchical models. In particular, it is determined that the E. coli and the yeast PINs are well represented by the stochastic and deterministic hierarchical network models respectively. These results suggest that the hierarchical network model is a better description for certain species' PINs. Furthermore, it is demonstrated that PINs are robust with respective to failure, attack, random rewiring and edge deletion perturbations.