bluuebunny/crossref_metadata_2025 huggingface dataset:
Field evaluation of the versatile aerosol concentration enrichment system (VACES) particle concentrator coupled to the rapid single‐particle mass spectrometer (RSMS‐3)
Authors: Zhao Y. , Bein K. J. , Wexler A. S. , Misra C. , Fine P. M. , Sioutas C.
Published: February 2005

A field evaluation of versatile aerosol concentration enrichment system (VACES) coupled to a rapid single‐particle mass spectrometer (RSMS‐3) was conducted as part of the U.S. Environmental Protection Agency Supersite program in Pittsburgh during March 2002. RSMS‐3 hit rate increases were measured, and possible particle composition changes introduced by the VACES were examined in the single‐particle mass spectra. The hit rates increased by 5–20 times at particle sizes ranging from 40 to 640 nm. VACES only enhances the hit rate by about a factor of 2 for large particle sizes because the RSMS‐3 flow rates for these particles did not match the optimum operating condition of VACES. During the 3 days of measurements most of the particles were a mixture of carbonaceous material and ammonium nitrate with a variation across the spectrum from particles that were mostly carbonaceous to particles that were mostly ammonium nitrate. Both ambient and concentrated carbonaceous and ammonium nitrate composition distributions were indistinguishable with RSMS‐3, suggesting that VACES introduces an insignificant artifact for those particles.
Hydrologic simulations in the Rhine basin driven by a regional climate model
Authors: Kleinn J. , Frei C. , Gurtz J. , Lüthi D. , Vidale P. L. , Schär C.
Published: February 2005

We describe and evaluate a model chain for studying streamflow responses to climate variations and anthropogenic climate change. The model chain was developed for the Rhine basin upstream of Cologne, a 145,000 km2 river basin in Central Europe north of the Alps. It encompasses a regional climate model (RCM) at grid spacings of 56 and 14 km, and a distributed hydrological model with a grid spacing of 1 km. The hydrological model is one‐way nested into the RCM through a downscaling interface, which introduces fine‐scale structures in the forcing data (i.e. temperature, precipitation, total net surface radiation, 10‐m wind speed, and relative humidity). Biases in precipitation and temperature are accounted for by catchment‐dependent but seasonally constant correction factors. Apart from these bias corrections, the hydrological model is forced by hourly RCM data. In the evaluation we compare a 5‐year integration driven by observed lateral boundary conditions (ECMWF reanalysis) against daily analysis of high‐density rain‐gauge data and streamflow data. The regional climate model is found to qualitatively reproduce the main mesoscale precipitation patterns and their seasonal evolution. Systematic biases are, however, found in the distribution of precipitation with topographic height in the Alpine region and at adjacent hill ranges. The RCM also reproduces intercatchment variations in the frequency distribution of daily precipitation. Simulated runoff resembles closely the mean annual cycle, and daily runoff agrees well with observations in timing and amplitude of runoff events for lowland gauges. Larger model errors are found for high‐altitude Alpine catchments. The 14‐km RCM provides much finer and more realistic precipitation fields compared to the 56‐km RCM, but these improvements did not have a significant impact on the skill of the hydrological model to simulate streamflow. The model chain was found to reproduce observed month‐to‐month variations of basin‐mean winter precipitation and streamflow with correlations between 0.85 and 0.95. This result provides confidence that the model chain is able to represent key processes related to streamflow variations in response to climate variations and climate change.
Evaluation of temperature and wind over Antarctica in a Regional Atmospheric Climate Model using 1 year of automatic weather station data and upper air observations
Authors: Reijmer C. H. , van Meijgaard E. , van den Broeke M. R.
Published: February 2005

The Regional Atmospheric Climate Model version 2 (RACMO2/ANT) is used to simulate the Antarctic atmosphere for the year 1998. The parameterizations of the physical processes in the model are taken from the European Centre for Medium‐Range Weather Forecasts (ECMWF) global model and adapted to better represent the specific conditions over the Antarctic continent. The snow albedo was increased by decreasing the rate of albedo change as a function of temperature. This results in a decrease, and an improvement, in near‐surface air temperatures in summer, especially over the Antarctic plateau. The surface roughness length for momentum was decreased. This increases the wind speed over the total atmospheric column. As a result, wind speeds better correspond to measurements, especially near the surface and in high wind speed areas. Comparison with ECMWF 40‐year reanalyses (ERA40) data show that RACMO2/ANT better reproduces the near‐surface conditions in terms of temperature and wind speed mainly due to the changes made to the physics description. Away from the surface, ERA40 better corresponds to observations due to the inclusion of the observed profiles in the reanalyses.
Impact of tree clearing on soil pH and nutrient availability in grazing systems of central Queensland, Australia
Authors: Sangha Kamaljit K. , Jalota Rajesh K. , Midmore David J.
Published: February 2005

In Queensland, land is cleared at high rates to develop pastures for enhanced production and the associated monetary gains. However, pasture production declines over time in cleared pastures until a new equilibrium is reached. The present study focussed on elucidating the reasons for decline in pasture production and finding the key soil properties that are affected due to clearing. Paired sites for cleared and uncleared pastures were selected to represent 3 dominant tree communities of the semi-arid tropics in central Queensland, i.e. Eucalyptus populnea, E. melanophloia, and Acacia harpophylla. The cleared pastures were chosen to represent 3 different durations of time since clearing (5, 11–13, and 33 years) to evaluate the temporal impact of clearing on soil properties. Various soil parameters were studied: macronutrients—available N (NH4+and NO3–), total N, and available P; micronutrients—Cu, Fe, Zn, and Mn; exchangeable cations—Ca, Mg, Na, and K (also macronutrients); pHw; and electrical conductivity. Of these, pHw showed a significant response to time of clearing for all 3 tree communities. Soil pHw increased significantly at cleared sites relative to uncleared (native woodland) pastures, and the increase was highly correlated with concentrations of exchangeable Ca, Mg, and Na. The change in soil pHw and exchangeable cations was more evident at >0.30 m soil depth. The increase in soil pHw in cleared pastures decreased the availability of soil nutrients for plant growth and, hence, pasture productivity. The interactions of different soil properties down the profile as a result of changes caused by clearing are important when interpreting the effects of clearing on soil properties.
The effect of ionic strength variation and anion competition on the development of nitrate accumulations in variable charge subsoils
Authors: Donn M. J. , Menzies N. W.
Published: February 2005

The leaching of N fertilisers has led to the formation of nitrate (NO3) accumulations in deep subsoils (>5 m depth) of the Johnstone River catchment. This paper outlines the chemical mechanism by which these NO3 accumulations are formed and maintained. This was achieved via a series of column experiments designed to investigate NO3 leaching in relation to the soil charge chemistry and the competition of anions for exchange sites. The presence of variable charge minerals has led to the formation positive surface charge within these profiles. An increase in the soil solution ionic strength accompanying the fertiliser leaching front acts to increase the positive (and negative) charge density, thus providing adsorption sites for NO3. A decrease in the soil solution ionic strength occurs after the fertiliser pulse moves past a point in the profile, due to dilution with incoming rainwater. Nitrate is then released from the exchange back into the soil solution, thus buffering the decrease in the soil solution ionic strength. Since NO3 was adsorbed throughout the profile in this experiment it does not effectively explain the situation occurring in the field. Previous observations of the sulfate (SO4) profile distribution indicated that large SO4 accumulations in the upper profile may influence the NO3 distribution through competition for adsorption sites. A subsequent experiment investigating the effect of SO4 additions on NO3 leaching showed that NO3 adsorption was minimal in the upper profile. Adsorption of NO3 did occur, though only in the region of the profile where SO4 occupancy was low, i.e. in the lower profile. Therefore, the formation of the NO3 accumulations is dependent on the variable charge mineralogy, the variation of charge density with soil solution ionic strength, and the effects of SO4 competition for adsorption sites.
Identification of combinatorial drug regimens for treatment of Huntington's disease using
Drosophila
Authors: Agrawal Namita , Pallos Judit , Slepko Natalia , Apostol Barbara L. , Bodai Laszlo , Chang Ling-Wen , Chiang Ann-Shyn , Thompson Leslie Michels , Marsh J. Lawrence
Published: February 2005

We explore the hypothesis that pathology of Huntington's disease involves multiple cellular mechanisms whose contributions to disease are incrementally additive or synergistic. We provide evidence that the photoreceptor neuron degeneration seen in flies expressing mutant human huntingtin correlates with widespread degenerative events in the
Drosophila
CNS. We use a
Drosophila
Huntington's disease model to establish dose regimens and protocols to assess the effectiveness of drug combinations used at low threshold concentrations. These proof of principle studies identify at least two potential combinatorial treatment options and illustrate a rapid and cost-effective paradigm for testing and optimizing combinatorial drug therapies while reducing side effects for patients with neurodegenerative disease. The potential for using prescreening in
Drosophila
to inform combinatorial therapies that are most likely to be effective for testing in mammals is discussed.
Predictors of dyadic friendship quality in adolescence
Authors: Cillessen Antonius H. N. , Jiang X. Lu , West Tessa V. , Laszkowski Dagmara K.
Published: February 2005

Five dimensions of friendship quality (conflict, closeness, companionship, helping, and security) were predicted from self-reports and peer reports of physical aggression, relational aggression, and prosocial behaviour, using the Actor-Partner Interdependence Model (Kenny & Acitelli, 2001). Participants were 224 adolescents aged 15–17 years (142 girls, 82 boys) who formed 112 unique same-sex best friend dyads. Significant actor and partner effects were found for both self-ratings and peer nominations of social behaviour and the five friendship qualities. Aggression was associated with self and partner perceptions of friendship conflict and low positive friendship qualities. Prosocial behaviour was associated with self and partner perceptions of positive friendship qualities and low conflict. The findings of this study were mostly consistent between male and female dyads. The importance of examining dependence due to dyads in peer relations research was discussed.
The Actor–Partner Interdependence Model: A model of bidirectional effects in developmental studies
Authors: Cook William L. , Kenny David A.
Published: February 2005

The actor–partner interdependence model (APIM) is a model of dyadic relationships that integrates a conceptual view of interdependence with the appropriate statistical techniques for measuring and testing it. In this article we present the APIM as a general, longitudinal model for measuring bidirectional effects in interpersonal relationships. We also present three different approaches to testing the model. The statistical analysis of the APIM is illustrated using longitudinal data on relationship specific attachment security from 203 mother–adolescent dyads. The results support the view that interpersonal influence on attachment security is bidirectional. Moreover, consistent with a hypothesis from attachment theory, the degree to which a child’s attachment security is influenced by his or her primary caregiver is found to diminish with age.
Involvement of the Actin Cytoskeleton and Homotypic Membrane Fusion in ER Dynamics inCaenorhabditis elegans
Authors: Poteryaev Dmitry , Squirrell Jayne M. , Campbell Jay M. , White John G. , Spang Anne
Published: February 2005

The endoplasmic reticulum (ER) is the major intracellular membrane system. The ER is essential for protein and lipid biosynthesis, transport of proteins along the secretory pathway, and calcium storage. Here, we describe our investigations into the dynamics and regulation of the ER in the early Caenorhabditis elegans embryo. Using a GFP fusion to the ER-resident signal peptidase SP12, we observed the morphological transitions of the ER through fertilization and the early cell-cycles in living embryos. These transitions were tightly coordinated with the division cycle: upon onset of mitosis, the ER formed structured sheets that redispersed at the initiation of cleavage. Although microtubules were not required for the transition of the ER between these different states, the actin cytoskeleton facilitated the dispersal of the ER at the end of mitosis. The ER had an asymmetric distribution in the early embryo, which was dependent on the establishment of polarity by the PAR proteins. The small GTPase ARF-1 played an essential role in the ER dynamics, although this function appeared to be unrelated to the role of ARF-1 in vesicular traffic. In addition, the ER-resident heat shock protein BiP and a homologue of the AAA ATPase Cdc48/p97 were found to be crucial for the ER transitions. Both proteins have been implicated in homotypic ER membrane fusion. We provide evidence that homotypic membrane fusion is required to form the sheet structure in the early embryo.
Plasticity of B Cell Receptor Internalization upon Conditional Depletion of Clathrin
Authors: Stoddart Angela , Jackson Antony P. , Brodsky Frances M.
Published: February 2005

B cell antigen receptor (BCR) association with lipid rafts, the actin cytoskeleton, and clathrin-coated pits influences B cell signaling and antigen presentation. Although all three cellular structures have been separately implicated in BCR internalization, the relationship between them has not been clearly defined. In this study, internalization pathways were characterized by specifically blocking each potential mechanism of internalization. BCR uptake was reduced by ∼70% in B cells conditionally deficient in clathrin heavy chain expression. Actin or raft antagonists were both able to block the residual, clathrin-independent BCR internalization. These agents also affected clathrin-dependent internalization, indicating that clathrin-coated pits, in concert with mechanisms dependent on rafts and actin, mediate the majority of BCR internalization. Clustering GM1gangliosides enhanced clathrin-independent BCR internalization, and this required actin. Thus, although rafts or actin independently did not mediate BCR internalization, they apparently cooperate to promote some internalization even in the absence of clathrin. Simultaneous inhibition of all BCR uptake pathways resulted in sustained tyrosine phosphorylation and activation of the extracellular signal-regulated kinase (ERK), strongly suggesting that downstream BCR signaling can occur without receptor translocation to endosomes and that internalization leads to signal attenuation.
Histopathology of carotid body in heroin addiction. Possible chemosensitive impairment
Authors: Porzionato A , Macchi V , Guidolin D , Parenti A , Ferrara S D , De Caro R
Published: February 2005

Aims : To perform a morphometric analysis of carotid bodies in opiate addicts.

Methods and results : Carotid bodies were sampled at autopsy from 35 subjects who died of heroin intoxication (mean age 26 years), and from eight young (22 years) and eight older subjects (66.5 years) who died of trauma. Sections were stained with haematoxylin–eosin, azan‐Mallory, and double‐labelling immunohistochemistry with antineuronal specific enolase and anti‐S100, to count type I and type II cells. Interlobular and intralobular connective tissue was increased both in the opiate cases (43.45 ± 6.79%, P < 0.001, and 13.34 ± 5.72%, P < 0.001, respectively) and older cases (46.67 ± 1.65%, P < 0.001, and 9.62 ± 2.11%, P < 0.05, respectively) compared with young cases (33.17 ± 6.41% and 4.33 ± 1.84%, respectively). The percentage of type II cells in the opiate cases (51.6 ± 7.3%, P < 0.001) and in the older controls (49.0 ± 7.2%, P < 0.01) was higher than in the young cases (37.9 ± 3.0%). Among type I cells, the light cell percentage in the opiate cases (65.85 ± 11%, P < 0.001) was reduced with respect to the two control groups (82.8 ± 5.34%, young; 81.62 ± 8.58%, older).

Conclusions : The increases in connective tissue and type II cells are similar to findings in ageing and chronic pulmonary disease, and may be ascribed to glomic hypoxia. A direct action of opiates should be taken into account for the decrease in light cells in heroin addiction. The histopathological changes in the carotid body, by impairing chemosensivity, may play a role in the fatal cardiorespiratory derangement of heroin addicts.
The distribution of cardiac macrophages in myocardial ischaemia and cardiomyopathy
Authors: Azzawi M , Kan S W , Hillier V , Yonan N , Hutchinson I V , Hasleton P S
Published: February 2005

Aims : Recent evidence has implicated the macrophage as an effector cell in the inflammatory processes in transplant rejection, as well as cardiac disease, including coronary atherosclerosis. Although the latter is a vascular disease, the entire myocardium is affected. We have previously demonstrated the presence and distribution of macrophages in the ‘normal’ human heart. In this paper the distribution of myocardial macrophages, in the various chambers of the failing human heart, from cases of coronary atheroma and cardiomyopathy undergoing heart transplantation is documented.

Methods and results : Tissue blocks were removed at specific sites taken from six cases with ischaemic heart disease (IHD) (four males, two females, age range 54–62 years), and four cases with idiopathic dilated cardiomyopathy (IDCM) (three males, one female, age range 18–49 years). These were compared with hearts from five cases of sudden death, unrelated to heart disease. Sections were stained with a CD68 pan macrophage marker. Positive cells were enumerated in 20 random fields. Results were analysed using a generalized linear modelling method using a Poisson distribution. Macrophages were identified within the interstitium and often close to blood vessels in all hearts. Macrophages from IHD hearts demonstrated the most intense staining and were often larger and more elongated than those found in ‘normal’ control hearts. Macrophages were also often degranulated and staining was diffuse in the interstitium. Overall, there were significantly more macrophages in most areas from IHD hearts than from IDCM hearts or control hearts (P < 0.001).

Conclusions : Significantly more macrophages were found in all four chambers in diseased hearts compared with controls. Macrophage numbers were higher in the atria than in ventricles in the diseased myocardium. This study suggests selective recruitment of macrophages into the atria in the disease states studied.
Retroviral Foxp3 gene transfer ameliorates liver granuloma pathology in Schistosoma mansoni infected mice
Authors: Singh Kameshwar P. , Gerard Herve C. , Hudson Alan P. , Reddy Thipparthi R. , Boros Dov L.
Published: February 2005

Schistosomiasis mansoni, a tropical helminthic disease, is caused by disseminated worm eggs that induce CD4+ T‐cell mediated granulomatous inflammation and fibrosis. T suppressor cell activity has been proposed as one of the mechanisms active in the down‐modulation of the murine disease during the chronic stage (16–20 weeks of the infection). In recent years a new category of the CD4+ CD25+ T regulatory (Treg) lymphocyte has been identified that maintains immune tolerance to self, and also functions in the regulation of parasite‐induced immunopathology. The Foxp3 gene which encodes the transcription factor Scurfin was found to be expressed by and required for the generation of CD4+ CD25+ T reg. At 8 weeks of the infection Foxp3 gene expression of splenocytes was similar to that of naïve mice, but increased fourfold by 16 weeks. In contrast, granulomatous livers at 8 and 16 weeks showed 10‐ and 30‐fold increases, respectively, in gene expression compared with normal liver. The percentage of granuloma CD4+ CD25+ T cells rose from 12% at 8 weeks to 88% at 16 weeks of the infection. Foxp3 expression was 3·5‐fold higher in the CD4+ CD25+ versus the CD4+ CD25– T cells in the 8 week infection granulomas. As a novel observation neuropilin‐1 membrane expression, a recently identified marker for Treg, was correlated with Foxp3 expression in the granuloma CD4+ CD25+ but not the CD25– cells. Co‐incubation with polyclonal stimulation of CD4+ CD25+ splenic cells with CD4+ CD25– cells suppressed proliferation of the latter. Retroviral transfer of the Foxp3 gene at the onset of granuloma formation enhanced fourfold Foxp3 expression in the granuloma CD4+ CD25+ T cells and strongly suppressed full granuloma development. Gene transfer also significantly enhanced transforming growth factor‐β, interferon‐γ and interleukin‐4 but not interleukin‐10 expression. It is concluded, that CD4+ CD25+, Foxp3+ Treg cells also regulate schistosome egg‐induced immunopathology.
Interleukin‐7 signalling is sufficient to phenotypically and functionally prime human CD4+ naïve T cells
Authors: Managlia Elizabeth Z. , Landay Alan , Al‐Harthi Lena
Published: February 2005

Interleukin‐7 (IL‐7) is produced by bone marrow and lymphoid stromal cells and is involved in the synthesis, survival and homeostasis of T cells. These attributes are the basis for current strategies to utilize IL‐7 as an immune modulator for several clinical conditions to replenish depleted T‐cell numbers. Because we had previously determined that IL‐7 can induce potent human immunodeficiency virus replication in the otherwise non‐permissive CD4+ naïve T‐cell compartment, we evaluated here the impact of IL‐7 on the phenotype and functional potential of naïve CD4+ T cells in an attempt to understand the mechanism of this induction. We demonstrate that IL‐7 mediated the up‐regulation of CD25, CD95 and human leucocyte antigen‐DR, while it did not alter the expression of CD45RO, CD69, CD40, or CD154. Examination of the cytokine profile of IL‐7‐treated naïve T cells using a Type1/Type2 Proteome Array indicated a remarkable IL‐7‐mediated induction of interferon‐γ production, while the other cytokines evaluated (IL‐2, IL‐12, tumour necrosis factor‐α, IL‐4, IL‐5, IL‐10 and IL‐13) were not affected. Intracellular staining of IL‐7‐treated naïve T cells for interferon‐γ verified the Proteome data. IL‐7 did not induce cell cycle proliferation of naïve CD4+ T cells, as evaluated by 7‐AAD/pyronin immunostaining and carboxyfluorescein diacetate succinimidyl ester dye tracking. IL‐7 treatment of naïve CD4+ T cells induced their ability to prime monocytes, as was indicated by induction of CD80 and CD86 expression on monocytes cocultured with IL‐7‐treated naïve CD4+ T cells. Collectively, these data indicate that IL‐7 signalling is sufficient to phenotypically and functionally prime human CD4+ naïve T cells independent of antigen stimulation.
Cross‐linking of neutrophil CD11b results in rapid cell surface expression of molecules required for antigen presentation and T‐cell activation
Authors: Sandilands Gavin P. , Ahmed Zubir , Perry Nicole , Davison Martin , Lupton Alison , Young Barbara
Published: February 2005

Recent studies suggest that neutrophils may play a role in antigen presentation. In support of this hypothesis it has been shown that these cells appear to contain cytoplasmic stores of molecules required for this function, i.e. major histocompatibility complex class II (DR) antigen, CD80 and CD86. In this study we have considered a mechanism for the translocation of these preformed molecules onto the cell surface which does not require active synthesis. Cross‐linking of the Mac‐1 molecule (CD18 + CD11b) was shown to result in rapid cell surface expression of CD80, CD86 and DR antigen on the surface of normal human peripheral blood neutrophils. A distinct subpopulation (approximately 20%) of neutrophils appeared to be enlarged and were found to express significantly elevated levels of these molecules on the cell surface following cross‐linking of CD11b when compared with control cells. The level of expression of CD80, CD86 and DR antigen on these large cells was comparable to, and in some cases greater than, the levels found expressed on the surface of monocytes obtained from the same donors. In addition, these cytoplasmic molecules were shown by confocal laser microscopy and by immunoelectron microscopy to be located within secretory vesicles. Following rapid translocation onto the cell surface, CD80 and CD86 appeared to be colocalized within large clusters reminiscent of the supramolecular antigen clusters previously found on conventional antigen‐presenting cells. These findings therefore lend further support for the hypothesis that neutrophils may have a role to play in antigen presentation and/or T‐cell activation.
Stoichiometric tapasin interactions in the catalysis of major histocompatibility complex class I molecule assembly
Authors: Bangia Naveen , Cresswell Peter
Published: February 2005

The assembly of major histocompatibility complex (MHC) class I molecules with their peptide ligands in the endoplasmic reticulum (ER) requires the assistance of many proteins that form a multimolecular assemblage termed the ‘peptide‐loading complex’. Tapasin is the central stabilizer of this complex, which also includes the transporter associated with antigen processing (TAP), MHC class I molecules, the ER chaperone, calreticulin, and the thiol‐oxidoreductase ERp57. In the present report, we investigated the requirements of these interactions for tapasin protein stability and MHC class I dissociation from the peptide‐loading complex. We established that tapasin is stable in the absence of either TAP or MHC class I interaction. In the absence of TAP, tapasin interaction with MHC class I molecules is long‐lived and results in the sequestration of existing tapasin molecules. In contrast, in TAP‐sufficient cells, tapasin is re‐utilized to interact with and facilitate the assembly of many MHC class I molecules sequentially. Furthermore, chemical cross‐linking has been utilized to characterize the interactions within this complex. We demonstrate that tapasin and MHC class I molecules exist in a 1 : 1 complex without evidence of higher‐order tapasin multimers. Together these studies shed light on the tapasin protein life cycle and how it functions in MHC class I assembly with peptide for presentation to CD8+ T cells.
Migration of polymorphonuclear leucocytes is influenced by dendritic cells
Authors: Scimone M. Lucila , Lutzky Viviana P. , Zittermann Sandra I. , Maffia Paulo , Jancic Carolina , Buzzola Fernanda , Issekutz Andrew C. , Chuluyan H. Eduardo
Published: February 2005

Dendritic cells (DCs) are the most potent antigen‐presenting cells and populate many tissues where they may participate in inflammatory reactions. The infiltration of polymorphonuclear leucocytes (PMNLs) into tissues is a prominent feature of inflammation. The mechanisms of PMNL recruitment depend on chemotactic factors and adhesion molecules expressed on endothelial cells. The aim of the present study was to determine whether DCs participate in the early recruitment of PMNLs. Dendritic cells derived from peripheral blood monocytes were used for this study. PMNLs incubated with culture supernatant (CS) from untreated or from tumour necrosis factor‐α (TNF‐α)‐treated (1 hr, 100 U/ml, 37°) monocyte‐derived DCs (moDCs) had increased surface expression of both CD11b and CD18. Moreover, both untreated and TNF‐α‐treated moDCs induced PMNL chemotaxis. By blocking CXCL8, CXCL5, CXCL7 and Pan GRO (CXCL1, CXCL2, CXCL3), we observed that CXCL8/interleukin‐8 might be the chemokine that induced the PMNL chemotactic activity in the CS of untreated and TNF‐α‐treated moDC. Furthermore, we investigated the regulation of CXCL8 production in moDCs by adhesion molecule engagement. Our data demonstrated that CD31, CD18, CD29 and CD49d participated in the adhesion of immature moDCs to endothelium. Moreover, engagement of domains 1–3 of CD31, but not of CD29 or CD18, decreased the production of CXCL8 by immature but not mature moDCs (which display lower CD31 levels than immature moDCs). Overall, these results suggest that DCs not only trigger a specific immune response, but also the innate immune response by recruiting PMNLs. Furthermore, our results also suggest that CXCL8 production by immature DCs might be regulated by signalling through CD31 during their migration through the vascular endothelium.
Interleukin‐10 (IL‐10) mediated suppression of IL‐12 production in RAW 264.7 cells also involves c‐rel transcription factor
Authors: Rahim Sheikh Showkat , Khan Nooruddin , Boddupalli Chandra Sekhar , Hasnain Seyed E. , Mukhopadhyay Sangita
Published: February 2005

Interleukin‐10 (IL‐10) is known to inhibit IL‐12 production in macrophages primarily at the transcriptional level with the involvement of p50 and p65 nuclear factor‐κB (NF‐κB). We demonstrate that the c‐rel transcription factor also plays a major role in IL‐10‐mediated IL‐12 suppression. Treatment of macrophages with recombinant IL‐10 inhibited nuclear c‐rel levels, whereas addition of neutralizing anti‐IL‐10 antibody up‐regulated both nuclear c‐rel levels and IL‐12 production by macrophages. Decreased nuclear c‐rel was associated with a reduction in phosphorylation of inhibitory kappa B alpha (IκBα) in the cytoplasm, indicating that IL‐10 prevents degradation of IκBα and the subsequent translocation of c‐rel into the nucleus. Treatment with leptomycin B, a known inhibitor of c‐rel at a concentration of 10 nm, when used with anti‐IL‐10 antibody, resulted in reduced expression of IL‐12. In a complementary experiment, in vitro transient expression of p65 NF‐κB could not rescue the inhibitory effect of IL‐10 on IL‐12 production, suggesting that NF‐κB alone was not sufficient to restore IL‐12 production during IL‐10 treatment. However, over‐expression of c‐rel resulted in IL‐12 restoration upon stimulation with lipopolysaccharide plus interferon‐γ during IL‐10 treatment. Our studies highlight the involvement of c‐rel in IL‐10‐mediated IL‐12 regulation.
Interferon sensitivity‐determining region of nonstructural region 5A of hepatitis C virus genotype 1b correlates with serum alanine aminotransferase levels in chronic infection
Authors: Yoshioka K. , Ito H. , Watanabe K. , Yano M. , Ishigami M. , Mizutani T. , Sasaki Y. , Goto H.
Published: February 2005

Summary.  The mutations in the interferon (IFN) sensitivity‐determining region (ISDR) of nonstructural region 5A (NS5A) of hepatitis C virus (HCV) have been correlated with response to IFN therapy. NS5A appears to disrupt a host antiviral pathway that plays a role in suppressing virus replication and protects hepatocytes from apoptosis. We assessed whether ISDR correlates with viral load and serum alanine aminotransferase (ALT) levels. Serum viral load and ALT levels were prospectively measured bimonthly by HCV core protein assay and monthly, respectively, for 22 months in 87 patients chronically infected with HCV genotype 1b. ISDR of HCV was directly sequenced from the products of reverse transcription and polymerase chain reaction of HCV RNA. Five patients had four or more substitutions (mutant type), 33 had 1–3 (intermediate type), and 49 had no substitutions (wild type) in ISDR. The numbers of substitutions in ISDR were inversely correlated with mean viral load over a 22‐month period (r = 0.292, P = 0.0060) and directly with mean serum ALT levels (r = 0.360, P = 0.0006). The numbers of substitutions in ISDR was significantly larger in the patients with changes of viral load more than fivefold during the 22 months (1.4 ± 2.4) than in those without changes (0.6 ± 0.8) (P = 0.0188). The present study demonstrates that the patients with more substitutions in ISDR had significantly higher serum ALT levels and smaller viral load. These results suggest that NS5A with more substitutions in ISDR may lose the ability to block host antiviral pathways and to protect hepatocytes from apoptosis.
Quantification of the relative levels of wild‐type and lamivudine‐resistant mutant virus in serum of HBV‐infected patients using microarray
Authors: Li Z. G. , Chen L. Y. , Huang J. , Qiao P. , Qiu J. M. , Wang S. Q.
Published: February 2005

Summary.  During the course of lamivudine administration in hepatitis B virus (HBV)‐infected patients, a dynamic development of the viral population in serum is observed. Total HBV level is initially reduced, then lamivudine‐resistant mutants appear, and finally, the viral level is increased. All methods of mutant detection so far described can only identify mutants in the serum, and cannot determine the proportion of those mutants. In this paper, we report the development of a novel technique that can quantify the relative proportion of mutants in serum utilizing gene microarray technology. Based on the nucleotide sequence at the loci of the mutations in lamivudine‐resistant HBV mutants, 28 probes were designed and dotted on glass film to prepare the oligonucleotide microarray. Ten standard curves were established by employing 15 reference plasmids as templates. Ten standard math functions were simulated, which allowed quantification of the proportion of mutants in the sample by measuring the value of fluorescent intensity on the microarray. By utilizing the standard math function, the relative proportion of two different mutation sequences in the mixed template could be detected with an error <10%. The HBV‐lamivudine oligonucleotide microarray is reliable to quantify the relative proportion of wild‐type HBV vs HBV mutants in patient's sera.
Evolution of a phage RuvC endonuclease for resolution of both Holliday and branched DNA junctions
Authors: Curtis Fiona A. , Reed Patricia , Sharples Gary J.
Published: February 2005

Resolution of Holliday junction recombination intermediates in most Gram‐negative bacteria is accomplished by the RuvC endonuclease acting in concert with the RuvAB branch migration machinery. Gram‐positive species, however, lack RuvC, with the exception of distantly related orthologues from bacteriophages infecting Lactococci and Streptococci. We have purified one of these proteins, 67RuvC, from Lactococcus lactis phage bIL67 and demonstrated that it functions as a Holliday structure resolvase. Differences in the sequence selectivity of resolution between 67RuvC and Escherichia coli RuvC were noted, although both enzymes prefer to cleave 3′ of thymidine residues. However, unlike its cellular counterpart, 67RuvC readily binds and cleaves a variety of branched DNA substrates in addition to Holliday junctions. Plasmids expressing 67RuvC induce chromosomal breaks, probably as a consequence of replication fork cleavage, and cannot be recovered from recombination‐defective E. coli strains. Despite these deleterious effects, 67RuvC constructs suppress the UV light sensitivity of ruvA, ruvAB and ruvABC mutant strains confirming that the phage protein mediates Holliday junction resolution in vivo. The characterization of 67RuvC offers a unique insight into how a Holliday junction‐specific resolvase can evolve into a debranching endonuclease tailored to the requirements of phage recombination.
Delineation and characterization of the actin nucleation and effector translocation activities of Salmonella SipC
Authors: Chang JiHoon , Chen Jue , Zhou Daoguo
Published: February 2005

Salmonella type III secreted SipC possesses dual functions: translocation of effectors and actin modulation. The biological significance of SipC's actin nucleation activity in Salmonella‐induced actin cytoskeleton rearrangements has not been studied. We report here the delineation of the actin nucleation activity from the effector translocation activity of SipC. Our data show that the central amino acid region (residues: 201–220) is essential for its actin nucleation activity and the C‐terminal amino acid region (321–409) is required for translocation of effectors. A SipC nucleation‐deficient mutant, which maintained its effector translocation activity, was obtained. This nucleation‐deficient mutant had significantly reduced ability to induce actin cytoskeleton rearrangements, resulting in lower bacterial invasion into HeLa cells. Contrary to a previous report, we found that the purified recombinant wild‐type SipC199‐409 protein is monomeric in solution by size exclusion chromatography coupled with multiangle laser light scattering assays (SEC‐LS). Our data established that the actin nucleation activity of SipC plays a vital role in Salmonella‐induced membrane ruffles and subsequent bacteria invasion.
The Ras and Rho GTPases genetically interact to co‐ordinately regulate cell polarity during development in Penicillium marneffei
Authors: Boyce Kylie J. , Hynes Michael J. , Andrianopoulos Alex
Published: February 2005

Ras and Rho GTPases have been examined in a wide variety of eukaryotes and play varied and often overlapping roles in cell polarization and development. Studies in Saccharomyces cerevisiae and mammalian cells have defined some of the central activities of these GTPases. However, these paradigms do not explain the role of these proteins in all eukaryotes. Unlike yeast, but like more complex eukaryotes, filamentous fungi have Rac‐like proteins in addition to Ras and Cdc42. To investigate the unique functions of these proteins and determine how they interact to co‐ordinately regulate morphogenesis during growth and development we undertook a genetic analysis of GTPase function by generating double mutants of the Rho GTPases cflA and cflB and the newly isolated Ras GTPase rasA from the dimorphic pathogenic fungus, Penicillium marneffei. P. marneffei growth at 25°C is as multinucleate, septate, branched hyphae which are capable of undergoing asexual development (conidiation), while at 37°C, uninucleate pathogenic yeast cells which divide by fission are produced. Here we show that RasA (Ras) acts upstream of CflA (Cdc42) to regulate germination of spores and polarized growth of both hyphal and yeast cells, while also exhibiting CflA‐independent activities. CflA (Cdc42) and CflB (Rac) co‐ordinately control hyphal cell polarization despite also having unique roles in regulating conidial germination and polarized growth of yeast cells (CflA) and polarized growth of conidiophore cell types and hyphal branching (CflB).
A che‐like signal transduction cascade involved in controlling flagella biosynthesis in Rhodospirillum centenum
Authors: Berleman James E. , Bauer Carl E.
Published: February 2005

Rhodospirillum centenum is a photosynthetic bacterium capable of undergoing swim cell to swarm cell differentiation that allows this species to be motile on both liquid and solid media. Previous experiments have demonstrated that the che1 operon is required for the control of chemotactic and phototactic behaviour of both swim and swarm cells. In this report, we analyse the function of a second che‐like gene cluster in R. centenum, the che2 gene cluster. In‐frame deletion mutants of cheW2, cheB2, cheR2, cheY2, and of the entire che2 operon, exhibit defects in swim and swarm cell motility. Analysis of these strains demonstrates that they are non‐motile, and that the non‐motile phenotype is resulting from reduced polar and lateral flagella synthesis. Additionally, mutations in mcp2, ORF204, cheA2 and ORF74 remain chemotacticly and phototacticly competent at both high and low growth temperatures. Mutations in these che2 genes result in elevated levels of flagellin proteins giving rise to a hyperflagellate phenotype. We propose a model in which R. centenum utilizes a che‐like signal transduction pathway (che2) for regulating flagellum synthesis in order to optimize swim cell‐swarm cell differentiation in response to changing environmental conditions.
HspR is a global negative regulator of heat shock gene expression in Deinococcus radiodurans
Authors: Schmid Amy K. , Howell Heather A. , Battista John R. , Peterson Scott N. , Lidstrom Mary E.
Published: February 2005

The HspR protein functions as a negative regulator of chaperone and protease gene expression in a diversity of bacteria. Here we have identified, cloned and deleted the Deinococcus radiodurans HspR homologue, DR0934. ΔhspR mutants exhibit moderate growth defects when shifted to mild heat shock temperatures, but are severely impaired for survival at 48°C. Using quantitative reverse transcription polymerase chain reaction and global transcriptional analysis, we have identified 14 genes that are derepressed in the absence of stress in the ΔhspR background, 11 of which encode predicted chaperones and proteases, including dnaKJgrpE, ftsH, lonB, hsp20 and clpB. Promoter mapping indicated that the transcription of these genes initiates from a promoter bearing a σ70‐type consensus, and that putative HspR binding sites (HAIR) were present in the 5′‐untranslated regions. Electrophoretic mobility shift assays indicated that HspR binds to these promoters at the HAIR site in vitro. These results strongly suggest that DR0934 encodes the HspR‐like global negative regulator of D. radiodurans that directly represses chaperone and protease gene expression by binding to the HAIR site in close proximity to promoter regions.
Changes in lipopolysaccharide structure induce the σE‐dependent response of Escherichia coli
Authors: Tam Christina , Missiakas Dominique
Published: February 2005

The envelope of Escherichia coli is composed of an asymmetric lipid bilayer containing lipopolysaccharide, phospholipid and outer membrane proteins (OMPs). Physical and chemical stresses impact on the integrity of the outer membrane envelope and trigger the σE‐dependent response, whereby E. coli activates the expression of genes that increase its capacity for folding OMPs and synthesizing lipopolysaccharide (LPS). While it has already been appreciated that misfolded OMPs induce the σE response, a role for LPS in activating this pathway was hitherto unknown. Here we show that ammonium metavandate (NH4VO3) induces multiple changes in E. coli LPS structure and activates the σE‐dependent response without altering OMP. One such NH4VO3‐mediated LPS decoration, the CrcA/PagP‐catalysed addition of palmitate to lipid A, appeared to be alone sufficient to activate transcription at σE‐dependent promoters. Furthermore, reduced acylation of LPS, caused by htrB or msbB mutations, also resulted in a constitutive expression of the σE regulon above wild‐type levels. Production of these aberrant outer membrane lipids did not noticeably affect the composition or the amount of OMPs. A model is proposed whereby structural intermediates of the LPS biosynthetic pathway or modified LPS molecules may function as signals that activate the σE response.
LmxMPK9, a mitogen‐activated protein kinase homologue affects flagellar length in Leishmania mexicana
Authors: Bengs Florian , Scholz Anne , Kuhn Daniela , Wiese Martin
Published: February 2005

Components of mitogen‐activated signal transduction pathways have been shown to be involved in flagellum biogenesis and maintenance. A mitogen‐activated protein kinase homologue, designated LmxMPK9 from Leishmania mexicana, has been recently identified in a homology screen and its mRNA found to be present in all life stages. Three different splice‐addition sites were used for mRNA maturation in trans‐splicing in the different life stages. However, here we show that LmxMPK9 protein is exclusively found in the promastigote stage. Recombinant expression of LmxMPK9 in Escherichia coli and kinase assays revealed a temperature optimum at 27°C, the optimal growth temperature for L. mexicana promastigotes, and a preference for manganese to promote substrate phosphorylation of myelin basic protein. A deletion mutant for the single‐copy gene revealed significantly elongated flagella, whereas overexpression led to a subpopulation with rather short to no flagella suggesting a role for LmxMPK9 in flagellar morphogenesis.
Net primary production and light use efficiency in a mixed coniferous forest in Sweden
Authors: LAGERGREN FREDERIK , EKLUNDH LARS , GRELLE ACHIM , LUNDBLAD MATTIAS , MÖLDER MEELIS , LANKREIJER HARRY , LINDROTH ANDERS
Published: February 2005

Simple light use efficiency (ɛ) models of net primary production (NPP) have recently been given great attention (NPP = ɛ × absorbed photosynthetically active radiation). The underlying relationships have, however, not been much studied on a time step less than a month. In this study daily NPP was estimated as the sum of net ecosystem exchange (NEE) and heterotrophic respiration (Rh) of a mixed pine and spruce forest in Sweden. NEE was measured by eddy correlation technique and Rh was estimated from measurements of forest floor respiration (Rf) and the root share of Rf. The total yearly NPP was on average 810 g C m−2 year−1 for 3 years and yearly ɛ was between 0.58 and 0.71 g C MJ−1, which is high in comparison with other studies. There was a seasonal trend in ɛ with a relatively constant level of approximately 0.90 g C MJ−1 from April to September Daily NPP did not increase for daily intercepted radiation above 6 MJ m−2 d−1, indicating that between‐years variation in NPP is not directly dependent on total Qi. The light was most efficiently used at an average daytime temperature of around 15 °C. At daytime vapour pressure deficit above 1400 Pa ɛ was reduced by approximately 50%.
On the relationship between isoprene emission and thermotolerance in Phragmites australis leaves exposed to high temperatures and during the recovery from a heat stress
Authors: VELIKOVA V. , LORETO F.
Published: February 2005

Thermotolerance induced by isoprene has been assessed during heat bursts but there is little information on the ability of endogenous isoprene to confer thermotolerance under naturally elevated temperature, on the interaction between isoprene‐induced thermotolerance and light stress, and on the persistence of this protection in leaves recovering at lower temperatures. Moderately high temperature treatment (38 °C for 1.5 h) reduced photosynthesis, stomatal conductance, and photochemical efficiency of photosystem II in isoprene‐emitting, but to a significantly lower extent than in isoprene‐inhibited Phragmites australis leaves. Isoprene inhibition and high temperature independently, as well as together, induced lipid peroxidation, increased level of H2O2, and increased catalase and peroxidase activities. However, leaves in which isoprene emission was previously inhibited developed stronger oxidative stress under high temperature with respect to isoprene‐emitting leaves. The heaviest photosynthetic stress was observed in isoprene‐inhibited leaves exposed to the brightest illumination (1500 µmol m−2 s−1) and, in general, there was also a clear additive effect of light excess on the formation of reactive oxygen species, antioxidant enzymes, and membrane damage. The increased thermotolerance capability of isoprene‐emitting leaves may be due to isoprene ability to stabilize membranes or to scavenge reactive oxygen species. Irrespective of the mechanism by which isoprene reduces thermal stress, isoprene‐emitting leaves are able to quickly recover after the stress. This may be an important feature for plants coping with frequent and transient temperature changes in nature.