bluuebunny/crossref_metadata_2025 huggingface dataset:
Examination of the Coordinate Effects ofPseudomonas aeruginosaExoS on Rac1
Authors: Rocha Claudia L. , Rucks Elizabeth A. , Vincent Deanne M. , Olson Joan C.
Published: August 2005

Exoenzyme S (ExoS) is a bifunctional toxin directly translocated into eukaryotic cells by thePseudomonas aeruginosatype III secretory (TTS) process. The amino-terminal GTPase-activating (GAP) activity and the carboxy-terminal ADP-ribosyltransferase (ADPRT) activity of ExoS have been found to target but exert opposite effects on the same low-molecular-weight G protein, Rac1. ExoS ADP-ribosylation of Rac1 is cell line dependent. In HT-29 human epithelial cells, where Rac1 is ADP-ribosylated by TTS-ExoS, Rac1 was activated and relocalized to the membrane fraction. Arg66 and Arg68 within the GTPase-binding region of Rac1 were identified as preferred sites of ExoS ADP-ribosylation. The modification of these residues by ExoS would be predicted to interfere with Rac1 inactivation and explain the increase in active Rac1 caused by ExoS ADPRT activity. Using ExoS-GAP and ADPRT mutants to examine the coordinate effects of the two domains on Rac1 function, limited effects of ExoS-GAP on Rac1 inactivation were evident in HT-29 cells. In J774A.1 macrophages, where Rac1 was not ADP-ribosylated, ExoS caused a decrease in the levels of active Rac1, and this decrease was linked to ExoS-GAP. Using immunofluorescence staining of Rac1 to understand the cellular basis for the targeting of ExoS ADPRT activity to Rac1, an inverse relationship was observed between Rac1 plasma membrane localization and Rac1 ADP-ribosylation. The results obtained from these studies have allowed the development of a model to explain the differential targeting and coordinate effects of ExoS GAP and ADPRT activity on Rac1 within the host cell.
Unusual Genetic Organization of a Functional Type I Protein Secretion System in
Neisseria meningitidis
Authors: Wooldridge Karl G. , Kizil Murat , Wells Damien B. , Ala'Aldeen Dlawer A. A.
Published: August 2005

Proteins secreted by
Neisseria meningitidis
are thought to play important roles in the pathogenesis of meningococcal disease. These proteins include the iron-repressible repeat-in-toxin (RTX) exoprotein FrpC. Related proteins in other pathogens are secreted via a type I secretion system (TOSS), but such a system has not been demonstrated in
N. meningitidis
. An in silico search of the group B meningococcal genome suggested the presence of a uniquely organized TOSS. Genes encoding homologs of the
Escherichia coli
HlyB (ATP-binding), HlyD (membrane fusion), and TolC (outer membrane channel) proteins were identified. In contrast to the cistronic organization of the secretion genes in most other
rtx
operons, the
hlyD
and
tolC
genes were adjacent but unlinked to
hlyB
; neither locus was part of an operon containing genes encoding putative TOSS substrates. Both loci were flanked by genes normally associated with mobile genetic elements. The three genes were shown to be expressed independently. Mutation at either locus resulted in an inability to secrete FrpC and a related protein, here called FrpC2. Successful complementation of these mutations at an ectopic site confirmed the observed phenotypes were caused by loss of function of the putative TOSS genes. We show that genes scattered in the meningococcal genome encode a functional TOSS required for secretion of the meningococcal RTX proteins.
Rate of Inversion of the
Salmonella enterica
Shufflon Regulates Expression of Invertible DNA
Authors: Tam Connie K. P. , Hackett Jim , Morris Christina
Published: August 2005

Salmonella enterica
serovar Typhi and some strains (Vi
+
) of serovar Dublin use type IVB pili to facilitate bacterial self-association, but only when the PilV proteins (potential minor pilus proteins) are not synthesized. Pilus-mediated self-association may be important in the pathogenesis of enteric fever. We have suggested that the rate of Rci-catalyzed inversion of DNA encoding the C-terminal portions of the PilV proteins controls PilV protein synthesis. This potentially represents a novel means of transcriptional control. Here, it is initially shown that DNA inversion per se is required for inhibition of gene expression from invertible DNA. Binding, without DNA scission, of Rci to its substrate sequences on DNA cannot explain the data obtained. Next, it is shown that inversion frequencies of
xylE
-encoding DNA, bracketed by Rci substrate sequences, may be modulated by changes in the 19-bp consensus sequences which are essential components of Rci substrate DNA. The affinity of Rci for these sequences affects inversion frequencies, so that a greater affinity is predictive of faster inversion, and therefore less synthesis of product encoded by invertible DNA. Inversion events may inhibit transcription of DNA from external promoters. In vivo, the frequency of Rci-mediated inversion is influenced by the extent of DNA supercoiling, with increasing levels of expression of invertible genes as novobiocin inhibits DNA supercoiling and thus Rci action. This inhibition of DNA supercoiling results in increased synthesis of PilV proteins as Rci activity decreases, and, in turn, bacterial self-association (particularly in serovar Dublin) decreases.
Helicobacter pylori-Specific CD4+T Cells Home to and Accumulate in the HumanHelicobacter pylori-Infected Gastric Mucosa
Authors: Lundgren Anna , Trollmo Christina , Edebo Anders , Svennerholm Ann-Mari , Lundin B. Samuel
Published: August 2005

Helicobacter pyloriinfects the stomach and duodenal mucosa. T cells are important components of theH. pylori-induced immune response, but little is currently known about how these cells are recruited to the infected mucosa. Here, we have characterized stomach and duodenal T cells isolated fromH. pylori-infected and noninfected subjects with regard to subtype, expression of homing and chemokine receptors, and in vitro reactivity toH. pyloriantigens. Higher numbers of CD4+but similar numbers of CD8+lamina propria T cells were isolated from stomach biopsies fromH. pylori-positive compared toH. pylori-negative individuals. CD4+T cells from infected stomach expressed increased levels of the homing receptor L-selectin and the chemokine receptor CCR4 compared to CD4+T cells from uninfected stomach. Infected stomach mucosa also contained increased levels of the CCR4 chemokine ligand MDC/CCL22. In contrast, comparable numbers of CD4+T cells with similar receptor expression were isolated from the duodenum ofH. pylori-positive andH. pylori-negative individuals. In vitro proliferation of mucosal T cells was strongly enhanced by the addition of interleukin-2 (IL-2) and IL-7 to the cell cultures. Using this approach,H. pylori-specific T-cell responses were detected in stomach CD4+T cells fromH. pylori-positive but notH. pylori-negative individuals. Duodenal T cells from only a few individuals responded toH. pyloristimulation, and the responsiveness was not restricted toH. pylori-positive individuals, suggesting limitedH. pylorispecificity in the duodenum and possible cross-reactivity with antigens from other bacteria in this compartment. In conclusion, these results suggest thatH. pylori-specific CD4+T cells preferentially home to and accumulate in the infected stomach and that L-selectin and CCR4/MDC are important for this recruitment.
Heteropentameric Cholera Toxin B Subunit Chimeric Molecules Genetically Fused to a Vaccine Antigen Induce Systemic and Mucosal Immune Responses: a Potential New Strategy To Target Recombinant Vaccine Antigens to Mucosal Immune Systems
Authors: Harakuni Tetsuya , Sugawa Hideki , Komesu Ai , Tadano Masayuki , Arakawa Takeshi
Published: August 2005

Noninvasive mucosal vaccines are attractive alternatives to parenteral vaccines. Although the conjugation of vaccine antigens with the B subunit of cholera toxin (CTB) is one of the most promising strategies for vaccine delivery to mucosal immune systems, the molecule cannot tolerate large-protein fusion, as it severely impairs pentamerization and loses affinity for GM1-ganglioside. Here we report a new strategy, in which steric hindrance between CTB-antigen fusion subunits is significantly reduced through the integration of unfused CTB “molecular buffers” into the pentamer unit, making them more efficiently self-assemble into biologically active pentamers. In addition, the chimeric protein took a compact configuration, becoming small enough to be secreted, and one-step affinity-purified proteins, when administered through a mucosal route, induced specific immune responses in mice. Since our results are not dependent on the use of a particular expression system or vaccine antigen, this strategy could be broadly applicable to bacterial enterotoxin-based vaccine design.
Characterization of Salivary Immunoglobulin A Responses in Children Heavily Exposed to the Oral Bacterium
Streptococcus mutans
: Influence of Specific Antigen Recognition in Infection
Authors: Nogueira Ruchele D. , Alves Alessandra C. , Napimoga Marcelo H. , Smith Daniel J. , Mattos-Graner Renata O.
Published: August 2005

The initial infection of children by
Streptococcus mutans
, the main pathogen of dental caries, depends on the ability of
S. mutans
to adhere and accumulate on tooth surfaces. These processes involve the adhesin antigen I/II (AgI/II), glucosyltransferases (GTF) and glucan-binding protein B (GbpB), each a target for anticaries vaccines. The salivary immunoglobulin A (IgA) antibody responses to
S. mutans
antigens (Ags) were characterized in 21 pairs of 5- to 13-month-old children. Pairs were constructed with one early
S. mutans
-infected and one noninfected child matched by age, racial background, number of teeth, and salivary levels of IgA. Specific salivary IgA antibody response and
S. mutans
infection levels were then measured during a 1-year follow-up. Robust responses to
S. mutans
were detected from 6 months of age. Salivary IgA antibody to AgI/II and GTF was commonly detected in salivas of all 42 children. However, GbpB-specific IgA antibody was seldom detected in the subset of infected children (38.1% at baseline). In contrast, most of the subset of noninfected children (76.2%) showed GbpB-reactive IgA antibody during the same period. Frequencies of GbpB responses increased with age, but differences in intensities of GbpB-IgA antibody reactions were sustained between the subsets. At baseline, GbpB-reactive IgA antibody accounted for at least half of the total salivary IgA
S. mutans
-reactive antibody in 33.3 and 9.5% of noninfected and infected children, respectively. This study provides evidence that a robust natural response to
S. mutans
Ags can be achieved by 1 year of age and that IgA antibody specificities may be critical in modulating initial
S. mutans
infection.
Identification of a Protein Subset of the Anthrax Spore Immunome in Humans Immunized with the Anthrax Vaccine Adsorbed Preparation
Authors: Kudva Indira T. , Griffin Robert W. , Garren Jeonifer M. , Calderwood Stephen B. , John Manohar
Published: August 2005

We
identified spore targets of Anthrax Vaccine Adsorbed (AVA)-induced
immunity in humans by screening recombinant clones of a previously
generated, limited genomic
Bacillus anthracis
Sterne
(pXO1
+
, pXO2
−
) expression library
of putative spore surface (spore-associated [SA]) proteins with pooled
sera from human adults immunized with AVA (immune sera), the anthrax
vaccine currently approved for use by humans in the United States. We
identified 69 clones that reacted specifically with pooled immune sera
but not with pooled sera obtained from the same individuals prior to
immunization. Positive clones expressed proteins previously identified
as localized on the anthrax spore surface, proteins highly expressed
during spore germination, orthologs of proteins of diverse pathogens
under investigation as drug targets, and orthologs of proteins
contributing to the virulence of both gram-positive and gram-negative
pathogens. Among the reactive clones identified by this immunological
screen was one expressing a 15.2-kDa hypothetical protein encoded by a
gene with no significant homology to sequences contained in databases.
Further studies are required to define the subset of SA proteins
identified in this study that contribute to the virulence of this
pathogen. We hypothesize that optimal delivery of a subset of SA
proteins identified by such studies to the immune system in combination
with protective antigen (PA), the principal immunogen in AVA, might
facilitate the development of defined, nonreactogenic, more-efficacious
PA-based anthrax vaccines. Future studies might also facilitate the
identification of SA proteins with potential to serve as targets for
drug design, spore inactivation, or spore detection
strategies.
Components of the
Legionella pneumophila
Flagellar Regulon Contribute to Multiple Virulence Traits, Including Lysosome Avoidance and Macrophage Death
Authors: Molofsky A. B. , Shetron-Rama L. M. , Swanson Michele S.
Published: August 2005

Legionella pneumophila
is a motile intracellular pathogen of macrophages and amoebae. When nutrients become scarce, the bacterium induces expression of transmission traits, some of which are dependent on the flagellar sigma factor FliA (σ
28
). To test how particular components of the
L. pneumophila
flagellar regulon contribute to virulence, we compared a
fliA
mutant with strains whose flagellar construction is disrupted at various stages. We find that
L. pneumophila
requires FliA to avoid lysosomal degradation in murine bone marrow-derived macrophages (BMM), to regulate production of a melanin-like pigment, and to regulate binding to the dye crystal violet, whereas motility, flagellar secretion, and external flagella or flagellin are dispensable for these activities. Thus, in addition to flagellar genes, the FliA sigma factor regulates an effector(s) or regulator(s) that contributes to other transmissive traits, notably inhibition of phagosome maturation. Whether or not the microbes produced flagellin, all nonmotile
L. pneumophila
mutants bound BMM less efficiently than the wild type, resulting in poor infectivity and a loss of contact-dependent death of BMM. Therefore, bacterial motility increases contact with host cells during infection, but flagellin is not an adhesin. When BMM contact by each nonmotile strain was promoted by centrifugation, all the mutants bound BMM similarly, but only those microbes that synthesized flagellin induced BMM death. Thus, the flagellar regulon equips the aquatic pathogen
L. pneumophila
to coordinate motility with multiple traits vital to virulence.
Pulmonary Interleukin-23 Gene Delivery Increases Local T-Cell Immunity and Controls Growth ofMycobacterium tuberculosisin the Lungs
Authors: Happel Kyle I. , Lockhart Euan A. , Mason Carol M. , Porretta Elizabeth , Keoshkerian Elizabeth , Odden Anthony R. , Nelson Steve , Ramsay Alistair J.
Published: August 2005

Interleukin-23 (IL-23) is a heterodimeric cytokine that shares IL-12 p40 but contains a unique p19 subunit similar to IL-12 p35. Previous studies indicate a greater importance for intact IL-12/23 p40 expression than IL-12 p35 for immunity againstMycobacterium tuberculosis, suggesting a role for IL-23 in host defense. The effects of IL-23 on the outcome of pulmonary infection withM. tuberculosishave not been described. Here, we show that local delivery of replication-defective adenovirus vectors encoding IL-23 (AdIL-23) greatly stimulated expression of both gamma interferon (IFN-γ) and IL-17 in lung tissues of otherwise normal mice. When given 72 h prior to infection withM. tuberculosis, AdIL-23 significantly reduced the bacterial burden at 14, 21, and 28 days. Markedly lower levels of lung inflammation were observed at 28 days than in control mice pretreated with control adenovirus (AdNull) or vehicle controls. AdIL-23 pretreatment resulted in increased numbers of CD4+CD25+activated T cells in lungs and draining lymph nodes compared to control groups and more CD4+T cells bearing surface memory markers in lung lymph nodes. IL-23 gene delivery also significantly enhanced host anti-mycobacterial T-cell responses, as shown by elevated levels of IFN-γ and IL-17 secreted in vitro following restimulation withM. tuberculosispurified protein derivative. Overall, our data show that transient IL-23 gene delivery in the lung is well tolerated, and they provide the initial demonstration that this factor controls mycobacterial growth while augmenting early pulmonary T-cell immunity.
Immunogenicity of Recombinant Protective Antigen and Efficacy against Aerosol Challenge with Anthrax
Authors: Williamson E. D. , Hodgson I. , Walker N. J. , Topping A. W. , Duchars M. G. , Mott J. M. , Estep J. , LeButt C. , Flick-Smith H. C. , Jones H. E. , Li H. , Quinn C. P.
Published: August 2005

Immunization with a recombinant form of the protective antigen (rPA) fromBacillus anthracishas been carried out with rhesus macaques. Rhesus macaques immunized with 25 μg or more ofB. subtilis-expressed rPA bound to alhydrogel had a significantly increased immunoglobulin G (IgG) response to rPA compared with macaques receiving the existing licensed vaccine from the United Kingdom (anthrax vaccine precipitated [AVP]), although the isotype profile was unchanged, with bias towards the IgG1 and IgG2 subclasses. Immune macaque sera from all immunized groups contained toxin-neutralizing antibody and recognized all the domains of PA. While the recognition of the N terminus of PA (domains 1 to 3) was predominant in macaques immunized with the existing vaccines (AVP and the U.S. vaccine anthrax vaccine adsorbed), macaques immunized with rPA recognized the N- and C-terminal domains of PA. Antiserum derived from immunized macaques protected macrophages in vitro against the cytotoxic effects of lethal toxin. Passive transfer of IgG purified from immune macaque serum into naive A/J mice conferred protection against challenge withB. anthracisin a dose-related manner. The protection conferred by passive transfer of 500 μg macaque IgG correlated significantly (P= 0.003;r= 0.4) with the titers of neutralizing antibody in donor macaques. Subsequently, a separate group of rhesus macaques immunized with 50 μg ofEscherichia coli-derived rPA adsorbed to alhydrogel was fully protected against a target dose of 200 50% lethal doses of aerosolizedB. anthracis. These data provide some preliminary evidence for the existence of immune correlates of protection against anthrax infection in rhesus macaques immunized with rPA.
Bordetella pertussis risA
, but Not
risS
, Is Required for Maximal Expression of Bvg-Repressed Genes
Authors: Stenson Trevor H. , Allen Andrew G. , al-Meer Jehan A. , Maskell Duncan , Peppler Mark S.
Published: August 2005

Expression of virulence determinants by
Bordetella pertussis
, the primary etiological agent of whooping cough, is regulated by the BvgAS two-component regulatory system. The role of a second two-component regulatory system, encoded by
risAS
, in this process is not defined. Here, we show that mutation of
B. pertussis risA
does not affect Bvg-activated genes or proteins. However, mutation of
risA
resulted in greatly diminished expression of Bvg-repressed antigens and decreased transcription of Bvg-repressed genes. In contrast, mutation of
risS
had no effect on the expression of Bvg-regulated molecules. Mutation of
risA
also resulted in decreased bacterial invasion in a HeLa cell model. However, decreased invasion could not be attributed to the decreased expression of Bvg-repressed products, suggesting that mutation of
risA
may affect the expression of a variety of genes. Unlike the
risAS
operons in
B. parapertussis
and
B. bronchiseptica
,
B. pertussis risS
is a pseudogene that encodes a truncated RisS sensor. Deletion of the intact part of the
B. pertussis risS
gene does not affect the expression of
risA
-dependent, Bvg-repressed genes. These observations suggest that RisA activation occurs through cross-regulation by a heterologous system.
Identification of Group A
Streptococcus
Antigenic Determinants Upregulated In Vivo
Authors: Salim Kowthar Y. , Cvitkovitch Dennis G. , Chang Peter , Bast Darrin J. , Handfield Martin , Hillman Jeffrey D. , de Azavedo Joyce C. S.
Published: August 2005

Group A
Streptococcus
(GAS) causes a range of diseases in humans, from mild noninvasive infections to severe invasive infections. The molecular basis for the varying severity of disease remains unclear. We identified genes expressed during invasive disease using in vivo-induced antigen technology (IVIAT), applied for the first time in a gram-positive organism. Convalescent-phase sera from patients with invasive disease were pooled, adsorbed against antigens derived from in vitro-grown GAS, and used to screen a GAS genomic expression library. A murine model of invasive GAS disease was included as an additional source of sera for screening. Sequencing DNA inserts from clones reactive with both human and mouse sera indicated 16 open reading frames with homology to genes involved in metabolic activity to genes of unknown function. Of these, seven genes were assessed for their differential expression by quantitative real-time PCR both in vivo, utilizing a murine model of invasive GAS disease, and in vitro at different time points of growth. Three gene products—a putative penicillin-binding protein 1A, a putative lipoprotein, and a conserved hypothetical protein homologous to a putative translation initiation inhibitor in
Vibrio vulnificus
—were upregulated in vivo, suggesting that these genes play a role during invasive disease.
Isolation and Partial Purification of Macrophage- and Dendritic Cell-Activating Components fromMycoplasma arthritidis: Association with Organism Virulence and Involvement with Toll-Like Receptor 2
Authors: Cole Barry C. , Mu Hong-Hua , Pennock Nathan D. , Hasebe Akira , Chan Fok V. , Washburn Leigh R. , Peltier Morgan R.
Published: August 2005

Mycoplasma arthritidisinduces toxicity, arthritis, and dermal necrosis in mice. Virulence factors include a superantigen and membrane adhesins and possibly also a bacteriophage component. Here we compare the biological properties of Triton X-114 extracts derived from avirulent and virulentM. arthritidisstrains. Macrophage cell lines and resident peritoneal macrophages were used to assess inflammatory potential as indicated by production of tumor necrosis factor alpha, interleukin-6, and/or nitric oxide. The activity resided exclusively within the hydrophobic detergent phase, was unaffected by heat treatment at 100°C for 30 min, and was resistant to proteinase K digestion, suggesting involvement of a lipopeptide. Contamination of extracts with endotoxin or superantigen was excluded. Extracts of the more virulent strain had higher activity than did those of the avirulent strain. Using CHO cells expressing Toll-like receptor 2 (TLR2) or TLR4, both with transfected CD14, we showed that extracts activated these cells via TLR2 but not by TLR4. Also, macrophages from C57BL/6 TLR2−/−mice failed to respond to the extracts, whereas those from TLR2+/+cells did respond. The preparations from the virulent strain ofM. arthritidiswere also more potent in activating dendritic cells, as evidenced by up-regulation of major histocompatibility complex class II, CD40, B7-1, and B7-2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent elution of gel slices revealed the presence of three active moieties which corresponded to molecular masses of approximately 24, 28, and 40 kDa. Three active components were also found by reverse-phase chromatography. We suggest that macrophage activation byM. arthritidiscould play a significant role in the inflammatory response induced in the host by this organism.
CpG Oligodeoxynucleotides Increase the Susceptibility of Normal Mice to Infection byCandida albicans
Authors: Ito Shuichi , Pedras-Vasconcelos Joao , Klinman Dennis M.
Published: August 2005

Synthetic oligodeoxynucleotides containing CpG motifs trigger an innate immune response that typically increases host resistance to infection. Yet CpG treatment reduces the resistance of normal mice toCandida albicansinfection. This effect is mediated by CpG-induced interleukin-12, indicating that CpG-dependent cytokine production may have adverse consequences for the host.
Airway Hyperresponsiveness to Methacholine in Chemical Warfare Victims
Authors: Mirsadraee M. , Attaran D. , Boskabady M.H. , Towhidi M.
Published: August 2005

Background: The lung is one of the most exposable organs to chemical warfare agents such as sulfur mustard gas. Pulmonary complications as a result of this gas range from severe bronchial stenosis to mild or no symptoms. Airway hyperresponsiveness (AHR) which is usually assessed as response to inhaled methacholine is the most characteristic feature of asthma. AHR is reported in chronic obstructive pulmonary disease patients and smokers, and may also show in chemical warfare victims. However, there are little reports regarding AHR in chemical warfare victims. Objective: Therefore, in this study, airway responsiveness to methacholine in victims of chemical warfare was examined. Methods: The threshold concentrations of inhaled methacholine required for a 20% change in forced expiratory flow in 1 s (FEV1; PC20) or a 35% change in specific airway conductance (PC35) were measured in 15 chemical war victims and 15 normal control subjects. Results: In 10 out of 15 chemical warfare victims (two thirds), PC20 and PC35 methacholine could be measured and subjects were called responders. AHR to methacholine in responder chemical war victims (PC20 = 0.41 and PC35 = 0.82 g/l) was significantly lower than in normal subjects (PC20 = 5.69 and PC35 = 4.60 g/l, p < 0.001 for both cases). There was a significant correlation between FEV1 and PC20 methacholine (r = 0.688, p < 0.001). The correlations between PC20 and PC35 were statistically significant as well (r = 0.856, p < 0.001). Conclusion: Results showed increased airway responsiveness of most chemical warfare victims to methacholine which correlated with the FEV1 value and which may be related to chronic airway inflammation or irreversible airway changes.
Inflammatory Response to Sputum Induction Measured by Exhaled Markers
Authors: Antczak Adam , Kharitonov Sergei A. , Montuschi Paolo , Gorski Pawel , Barnes Peter J.
Published: August 2005

Background: Sputum induction is increasingly used to study both cellular and biochemical composition of the airways. However, there is a significant rise in the percentage of neutrophils at 8 h after inhalation with hypertonic saline. Objective: The aim of this study was to assess whether markers of inflammation in exhaled air and exhaled air condensate change after sputum induction in normal and asthmatic subjects. Methods: We measured leukotriene B4 (LTB4) and a marker of oxidative stress, 8-isoprostane, (by enzyme immunoassay) in exhaled air condensate and exhaled nitric oxide (NO; by chemiluminescence analyzer) in 15 healthy subjects (8 females, mean age 35 ± 4 years, FEV1 97.4% predicted) and in 8 mild asthmatic subjects (5 males, mean age 34 ± 2 years, FEV1 70.5% predicted). Results: LTB4 was significantly higher compared with baseline at 6 h butdid not remain increased at 24 h after sputum induction (134.3 ± 30.15 and 75.4 ± 14.32 vs. 64.6 ± 11.6 pg/ml at baseline; p < 0.02 and p > 0.05, respectively) in healthy subjects. An inverse correlation between LTB4 and exhaled NO at 6 h after sputum induction was observed in healthy subjects (r = –0.66, p < 0.03). No increase in LTB4 levels was observed in asthmatic patients. Baseline 8-isoprostane levels were higher in asthmatic patients than in healthy subjects (47.3 ± 37.1 vs. 17.5 ± 8.8 pg/ml; p < 0.01). A trend towards increased levels of 8-isoprostane could be observed at 6 and 24 h after inhalation in healthy subjects (26.2 ± 3.7 and 26.7 ± 3.9 pg/ml; p = 0.09 and p = 0.07, respectively). In healthy subjects, exhaled NO was significantly higher compared with baseline at 6 h and remained increased 24 h after sputum induction (7.96 ± 3.5 vs. 5.61 ± 1.86 ppb; p < 0.01 and p < 0.05, respectively). Exhaled NO levels were increased in asthmatic patients but did not further increase after sputum induction. Conclusions: Sputum induction with hypertonic saline causes an inflammatory response which should be considered when using the technique to monitor airway inflammation.
Cytokine Imbalance in Infants Receiving Extracorporeal Membrane Oxygenation for Respiratory Failure
Authors: Mildner Reinout J. , Taub Nicholas , Vyas Julian R. , Killer Hillary M. , Firmin Richard K. , Field David J. , Kotecha Sailesh
Published: August 2005

Background: It is likely that the imbalance between the pro- and anti-inflammatory cytokines will determine the outcome in infants with severe respiratory failure receiving extracorporeal membrane oxygenation (ECMO). Aims: We determined if there was an imbalance between pro- and anti-inflammatory cytokines in serial bronchoalveolar lavage (BAL) fluid obtained from survivors and non-survivors of ECMO. Methods: We therefore measured the cellular changes and the molar ratios of pro-inflammatory and anti-inflammatory cytokines in serial BAL fluid obtained from survivors and non-survivors of ECMO. Fifteen infants surviving ECMO (median age 1 day, range 1–120) and 7 who did not (28 days, range 1–402) were studied. Results: In the lungs of survivors, the increased proportion of airway neutrophils at presentation decreased with time and was matched by a parallel increase in percent alveolar macrophages as the infants’ condition improved. The pro- and anti-inflammatory pulmonary cytokine ratios were static in the survivors. In the non-survivors, the ratio of tumour necrosis factor-α (TNF-α) against soluble TNF-receptor 1 (sTNF-R1) and soluble TNF receptor 2 (sTNF-R2) was increased at days 2–3 when compared to the survivors, but the molar ratio interleukin-1β (IL-1β)/soluble IL-1 receptor antagonist (sIL-1RA) was largely undetectable due to undetectable IL-1β. Conclusions: These data suggest that the infants who survive ECMO resolve their pulmonary inflammation and that in non-survivors the ratio of TNF-α against its receptor antagonists is increased and is associated with a poor outcome. Furthermore, this group of infants were unable to produce significant concentrations of IL-1β.
Galanin-Like Peptide Elicits a Robust Discharge of Growth Hormone in the Rhesus Monkey (Macaca mulatta)
Authors: Shahab Muhammad , Cunningham Matthew J. , Steiner Robert A. , Plant Tony M.
Published: August 2005

Galanin-like peptide (GALP) stimulates the release of gonadotropin-releasing hormone in rodent and primate species. The widespread distribution of GALP fibers in the hypothalamus suggests that this neuropeptide may influence hypophysiotropic factors that control other aspects of adenohypophysial function. Here we studied the effects of intracerebroventricular administration of GALP on serum levels of growth hormone (GH) and prolactin (PRL) in adult male monkeys. The animals (n = 5) were orchidectomized and implanted with testosterone-containing Silastic capsules to maintain the circulating testosterone levels (∼9 ng/ml) within the physiological range. The animals were implanted with an intracerebroventricular cannula and venous catheter for continuous access to the cerebroventricular and the venous circulation, respectively. GALP (500 µg), or vehicle alone, was administered as a bolus intracerebroventricular injection, and sequential blood samples were collected at 20-min intervals for 3 h before and after the injections. Within 20 min following GALP injection, the GH concentrations increased 3.5-fold, and a peak level (12.9 ± 2.7 ng/ml) was observed 40 min after injection. The GH levels remained elevated until 60 min after injection and thereafter declined to values similar to those observed at 0 min. The GH concentrations were not changed by vehicle alone. A decline in PRL levels was observed following GALP administration, with significantly reduced concentrations occurring between 60 and120 min following the injection of the neuropeptide. We conclude that in the monkey GALP is a potent secretagogue for GH and an inhibitor of PRL secretion and that GALP may, therefore, interact with the hypothalamic circuitry involved in the regulation of these pituitary hormones.
Targeting of a Novel Ca+
2
/Calmodulin-Dependent Protein Kinase II Is Essential for Extracellular Signal-Regulated Kinase–Mediated Signaling in Differentiated Smooth Muscle Cells
Authors: Marganski William A. , Gangopadhyay Samudra S. , Je Hyun-Dong , Gallant Cynthia , Morgan Kathleen G.
Published: August 2005

Subcellular targeting of kinases controls their activation and access to substrates. Although Ca
2+
/calmodulin-dependent protein kinase II (CaMKII) is known to regulate differentiated smooth muscle cell (dSMC) contractility, the importance of targeting in this regulation is not clear. The present study investigated the function in dSMCs of a novel variant of the γ isoform of CaMKII that contains a potential targeting sequence in its association domain (CaMKIIγ G-2). Antisense knockdown of CaMKIIγ G-2 inhibited extracellular signal-related kinase (ERK) activation, myosin phosphorylation, and contractile force in dSMCs. Confocal colocalization analysis revealed that in unstimulated dSMCs CaMKIIγ G-2 is bound to a cytoskeletal scaffold consisting of interconnected vimentin intermediate filaments and cytosolic dense bodies. On activation with a depolarizing stimulus, CaMKIIγ G-2 is released into the cytosol and subsequently targeted to cortical dense plaques. Comparison of phosphorylation and translocation time courses indicates that, after CaMKIIγ G-2 activation, and before CaMKIIγ G-2 translocation, vimentin is phosphorylated at a CaMKII-specific site. Differential centrifugation demonstrated that phosphorylation of vimentin in dSMCs is not sufficient to cause its disassembly, in contrast to results in cultured cells. Loading dSMCs with a decoy peptide containing the polyproline sequence within the association domain of CaMKIIγ G-2 inhibited targeting. Furthermore, prevention of CaMKIIγ G-2 targeting led to significant inhibition of ERK activation as well as contractility. Thus, for the first time, this study demonstrates the importance of CaMKII targeting in dSMC signaling and identifies a novel targeting function for the association domain in addition to its known role in oligomerization.
Associations of the Angiotensin II Type 1 Receptor A
1166
C and the Endothelial NO Synthase G
894
T Gene Polymorphisms With Silent Subcortical White Matter Lesions in Essential Hypertension
Authors: Henskens Léon H.G. , Kroon Abraham A. , van Boxtel Martin P.J. , Hofman Paul A.M. , de Leeuw Peter W.
Published: August 2005

Background and Purpose—

Silent white matter lesions (WMLs) may represent early target organ damage of the brain in patients with hypertension. Because these lesions may have a genetic background, we assessed the associations between polymorphisms of the renin-angiotensin system and the endothelial NO synthase (NOS3) genes and silent WMLs.

Methods—

Ninety-three hypertensive individuals were studied. MRI of the brain was performed to obtain estimates of the total volume of subcortical and the extent of periventricular WMLs. Patients were genotyped for the angiotensinogen (M
235
T), the angiotensin-converting enzyme (insertion/deletion [I/D]), the angiotensin II type 1 receptor (AGTR1 A
1166
C), and the NOS3 (G
894
T) genes. A linear regression model was used to assess the relationship of these gene polymorphisms with both subtypes of WMLs.

Results—

When adjusted for age, diabetes mellitus, and blood pressure, subcortical WML volume was lowest in the presence of 1 or 2 AGTR1 C alleles (unstandardized β, −38.8 [95% CI, −66.1 to −11.4] and −112.6 [CI, −188.9 to −36.4], respectively), whereas it was highest in the presence of an NOS3 T allele (3.1 [CI, 3.6 to 58.4]). No interaction between these polymorphisms on WMLs could be demonstrated. No associations were present with the other polymorphisms, either with subcortical or periventricular lesions.

Conclusions—

We found the AGTR1 A
1166
C as well as the NOS3 G
894
T polymorphisms to be associated with silent WMLs in the subcortical area.
Lightning-Initiation Locations as a Remote Sensing Tool of Large Thunderstorm Electric Field Vectors
Authors: Maggio Christopher , Coleman Lee , Marshall Thomas , Stolzenburg Maribeth , Stanley Mark , Hamlin Timothy , Krehbiel Paul , Rison William , Thomas Ron
Published: August 2005

The lightning data that are recorded with a three-dimensional lightning mapping array (LMA) are compared with data from an electric field change sensor (in this case a flat-plate antenna operated both as a “slow” and a “fast” antenna). The goal of these comparisons is to quantify any time difference that may exist between the initial responses of the two instruments to a lightning flash. The data consist of 136 flashes from two New Mexico thunderstorms. It is found that the initial radiation source detected by the LMA usually precedes the initial response of both the slow and fast antennas. In a small number of cases, the flat-plate antenna response precedes the initial LMA source, but by no more than 2 ms. The observations of such a close time coincidence suggest that the first LMA radiation source of each flash was located at or very near the flash-initiation point. Thus, the first LMA radiation source and the initial sequence of sources from a lightning flash can be used as remote sensing tools to give information about the magnitude of the electric field (relative to lightning-initiation thresholds) and the direction of the electric field at the initiation location.
Eddy-Induced Variability in a Transatlantic Section: Argo Observing System–Gyroscope 0302 Cruise Comparison
Authors: Vargas-Yáñez Manuel , Parrilla Gregorio , Lavín Alicia , Vélez-Belchí Pedro , González-Pola César , Hernández-Guerra Alonso
Published: August 2005

Ocean hydrological sections provide a very useful mean to study the ocean circulation as well as to determine water mass properties and to estimate fluxes. One basic method for their analysis is the spatial interpolation of data, obtained from a set of predefined stations, into a regular grid for contouring isolines and for further calculations. The shortest length scales that can be solved are limited by the distance between stations. Some of these scales, though resoluble by the sampling design, may be, with respect to time variability, shorter than the time that is needed to complete the section. This situation can produce a lack of synopticity in the obtained data, which is not usually addressed in oceanographic studies because the sequential repetition of oceanographic surveys is not an easy task. Here two samplings are compared—one by CTD- and another by Array for Real-Time Geostrophic Oceanography (Argo)-type profilers—of the same zonal section with a 5-day delay. The integral time scale for the mesoscale field is around 11 days, which implies that the mesoscale signal obtained from consecutive transmissions of the profilers are weakly correlated. The mesoscale field in a transatlantic section, which typically takes 20 days to be carried out, cannot be considered as synoptic.
An Incremental Remapping Transport Scheme on a Spherical Geodesic Grid
Authors: Lipscomb William H. , Ringler Todd D.
Published: August 2005

Weather and climate models contain equations for transporting conserved quantities such as the mass of air, water, ice, and associated tracers. Ideally, the numerical schemes used to solve these equations should be conservative, spatially accurate, and monotonicity-preserving. One such scheme is incremental remapping, previously developed for transport on quadrilateral grids. Here the incremental remapping scheme is reformulated for a spherical geodesic grid whose cells are hexagons and pentagons. The scheme is tested in a shallow-water model with both uniform and varying velocity fields. Solutions for standard shallow-water test cases 1, 2, and 5 are obtained with a centered scheme, a flux-corrected transport (FCT) scheme, and the remapping scheme. The three schemes are about equally accurate for transport of the height field. For tracer transport, remapping is far superior to the centered scheme, which produces large overshoots, and is generally smoother and more accurate than FCT. Remapping has a high startup cost associated with geometry calculations but is nearly twice as fast as FCT for each added tracer. As a result, remapping is cheaper than FCT for transport of more than about seven tracers.
Academic Glossolalia? Pentecostal Scholarship, Multi-Disciplinarity, and the Science-Religion Conversation
Authors: Yong Amos
Published: August 2005

As more and more Pentecostal institutions of higher education are being transformed from liberal arts colleges to universities, an increasing number of degrees in the social and natural sciences are being offered. At the same time, Pentecostals working and teaching in science and religion departments have not been engaged in the science-and-religion conversation in any measurable way. This essay attempts to chart the prospects for such an engagement by way of entering into dialogue from a Pentecostal perspective with three recent publications. Throughout, the importance and necessity for Pentecostal presence in the science-and-religion discussion is emphasized, especially with an eye toward revitalizing Pentecostal education, scholarship, and praxis for life in the twenty-first century.
Effective therapy of human lymphoma xenografts with a novel recombinant ribonuclease/anti-CD74 humanized IgG4 antibody immunotoxin
Authors: Chang Chien-Hsing , Sapra Puja , Vanama Sailaja S. , Hansen Hans J. , Horak Ivan D. , Goldenberg David M.
Published: August 2005

Ranpirnase (Rap) is a cytotoxic ribonuclease (RNase) isolated from frog oocytes. Here we describe high antitumor activity of a novel immunotoxin, 2L-Rap-hLL1-γ4P, composed of 2 Rap molecules, each fused to the N terminus of the light chain of hLL1, an internalizing anti-CD74 humanized antibody. To reduce unwanted side effects, the constant region of hLL1 was changed from γ1 to γ4 and further to γ4P by replacing serine228 to proline to prevent the formation of a half immunoglobulin G (IgG) common for IgG4. In vitro, 2L-Rap-hLL1-γ4P retained RNase activity, specific binding to CD74, and was significantly more potent against CD74+ cell lines (Daudi, Raji, and MC/CAR) than naked hLL1. In vivo, the pharmacokinetic profile of 2L-Rap-hLL1-γ4P was similar to that of naked hLL1. The maximum tolerated dose of 2L-Rap-hLL1-γ4P in severe combined immunodeficient mice (SCID) or BALB/c mice was 50 μg per mouse. In Raji and Daudi Burkitt lymphoma xenograft models, treatment with a single 5 to 50 μg dose of 2L-Rap-hLL1-γ4P, given as early or delayed treatment, resulted in cures of most animals. Treatment with 2L-Rap-hLL1-γ4P was significantly better than all controls, including saline, naked hLL1, and nonspecific immunotoxin. In conclusion, 2L-Rap-hLL1-γ4P demonstrated excellent in vitro and in vivo efficacy and thus merits further consideration as a therapeutic for CD74+ tumors.
Soluble CD30 serum levels before and after treatment with α-interferon in patients with chronic hepatitis C
Authors: Di Cesare Enrico , Spadaro Aldo , Ajello Antonino , Ferraù Oscar , Alessi Nunziata , Luigiano Carmelo , Melluso Rossana , Freni Maria A.
Published: August 2005

It has been suggested that soluble CD30 (sCD30) serum levels in chronic hepatitis C are correlated with the activity of the disease and with the outcome of interferon (IFN) treatment. In this study, sCD30 serum levels in 25 patients with chronic hepatitis C, before and after treatment with IFN-2α, were measured. A total of 20 healthy subjects were used as controls. High sCD30 levels in serum were found in 36% of patients and in 5% of controls. In patients with sCD30 levels above or within the normal range, no significant differences in age, gender, serum transaminases and histology activity index were found. In relation to IFN treatment, only responder patients had serum sCD30 higher than controls, although the difference between responders and non-responders was not significant. No changes from baseline values were observed after treatment. Although high, sCD30 serum levels in chronic hepatitis C are not correlated with the disease activity, are not affected by IFN treatment and are not predictors of response to IFN treatment.
Reference materials (RMs) for analysis of the human factor II (prothrombin) gene G20210A mutation
Authors: Klein Christoph L. , Márki-Zay János , Corbisier Philippe , Gancberg David , Cooper Susan , Gemmati Donato , Halbmayer Walter-Michael , Kitchen Steve , Melegh Béla , Neumaier Michael , Oldenburg Johannes , Leibundgut Elisabeth Oppliger , Reitsma Pieter H. , Rieger Sandra , Schimmel Heinz G. , Spannagl Michael , Tordai Attilia , Tosetto Alberto , Visvikis Sophie , Zadro Renata , Mannhalter Christine
Published: August 2005

The Scientific Committee of Molecular Biology Techniques (C-MBT) in Clinical Chemistry of the IFCC has initiated a joint project in co-operation with the European Commission, Joint Research Centre, Institute of Reference Materials and Measurements to develop and produce plasmid-type reference materials (RMs) for the analysis of the human prothrombin gene G20210A mutation. Although DNA tests have a high impact on clinical decision-making and the number of tests performed in diagnostic laboratories is high, issues of quality and quality assurance exist, and currently only a few RMs for clinical genetic testing are available. A gene fragment chosen was produced that spans all primer annealing sites published to date. Both the wild-type and mutant alleles of this gene fragment were cloned into a pUC18 plasmid and two plasmid RMs were produced. In addition, a mixture of both plasmids was produced to mimic the heterozygous genotype. The present study describes the performance of these reference materials in a commutability study, in which they were tested by nine different methods in 13 expert laboratories. This series of plasmid RMs are, to the best of our knowledge, the first plasmid-type clinical genetic RMs introduced worldwide.
Induction of Differentiation of Embryonic Stem Cells into Insulin-Secreting Cells by Fetal Soluble Factors
Authors: Vaca Pilar , Martín Franz , Vegara-Meseguer Josefina M. , Rovira Juan M. , Berná Genoveva , Soria Bernat
Published: August 2005

Cell signals produced during pancreas embryogenesis regulate pancreatic differentiation. We show that the developing pancreas releases soluble factors responsible for in vitro endocrine pancreatic differentiation from embryonic stem cells (ESCs). A mouse D3 ESC line was transfected with a human insulin promoter/βgeo/phosphoglycerate kinase–hygromycin-resistant construct. To direct differentiation, cells were cultured for 7 days to form embryoid bodies and then plated for an additional 7 days. During this 14-day period, besides eliminating leukemia inhibitory factor, cells were cultured in low serum concentration with the addition of conditioned media from embryonic day–16.5 pancreatic buds. Islet cell differentiation was studied by the following: (a) X-gal staining after neomycin selection, (b) BrdU (bro-modeoxyuridine) studies, (c) simple and double immunohistochemistry for insulin, C-peptide, and glucose transporter 2 (Glut-2), (d) reverse transcription–polymerase chain reaction for insulin and pancreas duodenum homeobox 1 (PDX-1), (e) insulin and C-peptide content and secretion assays, (f) intraperitoneal glucose tolerance test, (g) electrophysiology (patch-clamp studies in inside-out configuration), and (h) transplantation of differentiated cells under the kidney capsule of streptozotocin-diabetic mice. The differentiated ESCs showed the following: changes in the mRNA levels of insulin and PDX-1; coexpression of insulin, C-peptide, and Glut-2; glucose and tolbutamide-dependent insulin and C-peptide release; K-channel activity regulated by ATP; and normalization of blood glucose levels after transplantation into diabetic mice and hyperglycemia after graft removal. In this study, we establish a battery of techniques that could be used together to properly characterize islet cell differentiation. Moreover, identification of factors released by the developing pancreas may be instrumental in engineering β cells from stem cells.