Leukotrienes Target F-actin/Cofilin-1 to Enhance Alveolar Macrophage Anti-fungal Activity

Candida albicans is the most common opportunistic fungal pathogen and causes local and systemic disease in immunocompromised patients. Alveolar macrophages (AMs) are pivotal for the clearance of C. albicans from the lung. Activated AMs secrete 5-lipoxygenase-derived leukotrienes (LTs), which in turn enhance phagocytosis and microbicidal activity against a diverse array of pathogens. Our aim was to investigate the role of LTB(4) and LTD(4) in AM antimicrobial functions against C. albicans and the signaling pathways involved. Pharmacologic and genetic inhibition of LT biosynthesis as well as receptor antagonism reduced phagocytosis of C. albicans when compared with untreated or WT controls. Conversely, exogenous LTs of both classes augmented base-line C. albicans phagocytosis by AMs. Although LTB(4) enhanced mainly mannose receptor-dependent fungal ingestion, LTD(4) enhanced mainly dectin-1 receptor-mediated phagocytosis. LT enhancement of yeast ingestion was dependent on protein kinase C-delta (PKC delta) and PI3K but not PKC alpha and MAPK activation. Both LTs reduced activation of cofilin-1, whereas they enhanced total cellular F-actin; however, LTB(4) accomplished this through the activation of LIM kinases (LIMKs) 1 and 2, whereas LTD(4) did so exclusively via LIMK-2. Finally, both exogenous LTB(4) and LTD(4) enhanced AM fungicidal activity in an NADPH oxidase-dependent manner. Our data identify LTB(4) and LTD(4) as key mediators of innate immunity against C. albicans, which act by both distinct and conserved signaling mechanisms to enhance multiple antimicrobial functions of AMs.

Connective tissue mast cells are the target of formaldehyde to induce tracheal hyperresponsiveness in rats: Putative role of leukotriene B(4) and nitric oxide

Formaldehyde (FA) exposure induces upper airways irritation and respiratory abnormalities, but its mechanisms are not understood. Since mast cells are widely distributed in the airways, we hypothesized that FA might modify the airways reactivity by mechanism involving their activation. Tracheal rings of rats were incubated with Dulbecco`s modified medium culture containing FA (0.1 ppm) in 96-well plastic microplates in a humid atmosphere. After 30 min, 6 h, and 24-72 h, the rings were suspended in an organ bath and dose-response curve to methacholine (MCh) were determined. incubation with FA caused a transient tracheal hyperresponsiveness to MCh that was independent from tracheal epithelium integrity. Connective tissue mast cell depletion caused by compound 48/80 or mast cell activation by the allergic reaction, before exposure of tracheal rings to FA prevented the increased responsiveness to MCh. LTB(4) concentrations were increased in the culture medium of tracheas incubated with FA for 48 h, whereas the LTB(4)-receptor antagonist MK886 (1 mu M) added before FA exposure rendered the tracheal rings normoreactive to MCh. In addition, FA exposure did not cause hyperresponsiveness in tracheal segments incubated with L-arginine (1 mu M). We suggest that airway connective tissue mast cells constitute the target and may provide the increased LTB(4) generation as well as an elevated consumption of NO leading to tracheal hyperresponsiveness to MCh. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Nanostructured europium oxide thin films deposited by pulsed laser ablation of a metallic target in a He buffer atmosphere

Nanostrucured europium oxide and hydroxide films were obtained by pulsed Nd:YAG (532 nm) laser ablation of a europium metallic target, in the presence of a 1 mbar helium buffer atmosphere. Both the produced film and the ambient plasma were characterized. The plasma was monitored by an electrostatic probe, for plume expansion in vacuum or in the presence of the buffer atmosphere. The time evolution of the ion saturation current was obtained for several probe to substrate distances. The results show the splitting of the plume into two velocity groups, being the lower velocity profile associated with metal cluster formation within the plume. The films were obtained in the presence of helium atmosphere, for several target-to-substrate distances. They were analyzed by Rutherford backscattering spectrometry, x-ray diffraction, and atomic force microscopy, for as-deposited and 600 degrees C treated-in-air samples. The results show that the as-deposited samples are amorphous and have chemical composition compatible with europium hydroxide. The thermally treated samples show x-ray diffraction peaks of Eu(2)O(3), with chemical composition showing excess oxygen. Film nanostructuring was shown to be strongly correlated with cluster formation, as shown by velocity splitting in probe current versus time plots. (C) 2010 American Vacuum Society. [DOI: 10.1116/1.3457784]

Molecular characterization of Arabidopsis thaliana PUF proteins - binding specificity and target candidates

PUF proteins regulate both stability and translation through sequence-specific binding to the 3` UTR of target mRNA transcripts. Binding is mediated by a conserved PUF domain, which contains eight repeats of approximately 36 amino acids each. Found in all eukaryotes, they have been related to several developmental processes. Analysis of the 25 Arabidopsis Pumilio (APUM) proteins presenting PUF repeats reveals that 12 (APUM-1 to APUM-12) have a PUF domain with 50-75% similarity to the Drosophila PUF domain. Through three-hybrid assays, we show that APUM-1 to APUM-6 can bind specifically to the Nanos response element sequence recognized by Drosophila Pumilio. Using an Arabidopsis RNA library in a three-hybrid screening, we were able to identify an APUM-binding consensus sequence. Computational analysis allowed us to identify the APUM-binding element within the 3` UTR in many Arabidopsis transcripts, even in important mRNAs related to shoot stem cell maintenance. We demonstrate that APUM-1 to APUM-6 are able to bind specifically to APUM-binding elements in the 3` UTR of WUSCHEL, CLAVATA-1, PINHEAD/ZWILLE and FASCIATA-2 transcripts. The results obtained in the present study indicate that the APUM proteins may act as regulators in Arabidopsis through an evolutionarily conserved mechanism, which may open up a new approach for investigating mRNA regulation in plants.

Argininosuccinate Synthetase Is a Functional Target for a Snake Venom Anti-hypertensive Peptide ROLE IN ARGININE AND NITRIC OXIDE PRODUCTION

Bj-BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that Bj-BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological effect independent of angiotensin-converting enzyme inhibition. Therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for Bj-BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-dependent manner. AsS is recognized as an important player of the citrulline-NO cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of Bj-BPP-10c and AsS was evidenced by the following effects promoted by the peptide: (i) increase of NO metabolite production in human umbilical vein endothelial cell culture and of arginine in human embryonic kidney cells and (ii) increase of arginine plasma concentration in SHR. Moreover, alpha-methyl-DL-aspartic acid, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of Bj-BPP-10c in SHR. Taken together, these results suggest that AsS plays a role in the anti-hypertensive action of Bj-BPP-10c. Therefore, we propose the activation of AsS as a new mechanism for the anti-hypertensive effect of Bj-BPP-10c in SHR and AsS as a novel target for the therapy of hypertension-related diseases.

Under flow impedimetric measurements using magnetic particles for label-free detection affinity target

The necessity to adapt sensors based on electrochemical techniques for high throughput analysis control increases the interest to develop new analytical systems able to perform measurements under buffer now. In this report we explored the possibility of employing a new system to make impedimetric measurements to detect the interaction between proteins and small molecules. The well-known biotin-streptavidin interaction was adopted to evaluate the proposed assembly. This system allows us to perform experiments under flow. Magnetic beads functionalized with streptavidin were used and first characterized using AFM and FTIR. Non-faradic impedance spectroscopy allowed the detection of the biotin-streptavidin interaction. Using our new system and under a flow of PBS buffer, 5 10-5 M of biotin was detected with a stable signal. (c) 2007 Elsevier B.V. All rights reserved.

Oroidin Inhibits the Activity of the Multidrug Resistance Target Pdr5p from Yeast Plasma Membranes

Oroidin was isolated from the marine sponge Agelas sventres and inhibited the activity and function of Pdr5p, an enzyme responsible for the multidrug resistance phenotype in Saccharomyces cerevisiae. This compound may help in the development of new drugs that reverse this dangerous phenotype of pathogenic yeast and fungi.

Integration of Ligand- and Target-Based Virtual Screening for the Discovery of Cruzain Inhibitors

A myriad of methods are available for virtual screening of small organic compound databases. In this study we have successfully applied a quantitative model of consensus measurements, using a combination of 3D similarity searches (ROCS and EON), Hologram Quantitative Structure Activity Relationships (HQSAR) and docking (FRED, FlexX, Glide and AutoDock Vina), to retrieve cruzain inhibitors from collected databases. All methods were assessed individually and then combined in a Ligand-Based Virtual Screening (LBVS) and Target-Based Virtual Screening (TBVS) consensus scoring, using Receiving Operating Characteristic (ROC) curves to evaluate their performance. Three consensus strategies were used: scaled-rank-by-number, rank-by-rank and rank-by-vote, with the most thriving the scaled-rank-by-number strategy, considering that the stiff ROC curve appeared to be satisfactory in every way to indicate a higher enrichment power at early retrieval of active compounds from the database. The ligand-based method provided access to a robust and predictive HQSAR model that was developed to show superior discrimination between active and inactive compounds, which was also better than ROCS and EON procedures. Overall, the integration of fast computational techniques based on ligand and target structures resulted in a more efficient retrieval of cruzain inhibitors with desired pharmacological profiles that may be useful to advance the discovery of new trypanocidal agents.

CDK9 a potential target for drug development

The family of Cyclin-Dependent Kinases (CDKs) can be subdivided into two major functional groups based on their roles in cell cycle and/or transcriptional control. CDK9 is the catalytic subunit of positive transcription elongation factor b (P-TEFb). CDK9 is the kinase of the TAK complex (Tat-associated kinase complex), and binds to Tat protein of HIV, suggesting a possible role for CDK9 in AIDS progression. CDK9 complexed with its regulatory partner cyclin T1, serves as a cellular mediator of the transactivation function of the HIV Tat protein. P-TEFb is responsible for the phosphorylation of the carboxyl-terminal domain of RNA Pol II, resulting in stimulation of transcription. Furthermore, the complexes containing CDK9 induce the differentiation in distinct tissue. The CDK9/cyclin T1 complex is expressed at higher level in more differentiated primary neuroectodermal and neuroblastoma tumors, showing a correlation between the kinase expression and tumor differentiation grade. This may have clinical and therapeutical implications for these tumor types. Among the CDK inhibitors two have shown to be effective against CDK9: Roscovitine and Flavopiridol. These two inhibitors prevented the replication of human immunodeficiency virus (HIV) type 1 by blocking Tat transactivation of the HIV type 1 promoter. These compounds inhibit CDKs by binding to the catalytic domain in place of ATP, preventing transfer of a phosphate group to the substrate. More sensitive therapeutic agents of CDK9 can be designed, and structural studies can add information in the understanding of this kinase. The major features related to CDK9 inhibition will be reviewed in this article.

Oscar Wilde’s “The Happy Prince” : The Hidden Messages and the Debate on the Target Audience

Oscar Wilde’s fairytales have been read to children for more than a century. Nevertheless, since the time of their publication in 1888 and 1891, the target audience of The Happy Prince and Other Tales and A House of Pomegranates have been the concern of critics. Delving into the context behind the rich and colourful imagery, one can find implications of homosexuality, the Paterian aesthetic and religious connotations. According to Carol Tattersall, The Happy Prince and Other Tales successfully mislead the public that it is innocent of any intention to undermine established standards of living or writing. Tattersall’s argument is based on comparing the first collection to Wilde’s second, A House of Pomegranates, which was perceived as “offensive and immoral” (136). On the other hand, William Butler Yeats states in his introduction to The Complete Works of Oscar Wilde that overall the reviewers of The Happy Prince and Other Tales were hostile because of Wilde’s aesthetic views (ixxvi). But Yeats overlooks the fact that Wilde was very pleased and proud, dashing notes to friends and reviewers and signing copies to many people (Tattersall 129). In general, the reception of Wilde’s first collection was more positive than that of the second because it was milder and more subtle in its controversial themes.

English as the target language : A literature study on teachers’ and L2 learners’ language use in the upper elementary classroom

Even though English is a subject where Swedish pupils do well compared to pupils in other countries, research indicates that pupils are not always motivated to learn in the English classroom. Therefore, the aim of this study is to find research relating to the use of the target language in classrooms for pupils at the upper elementary level, particularly language learners in Sweden. The focus of this thesis is to find out what benefits and challenges accompany the use of the target language during English lessons, as well as what pupils’ opinions are on the consistent use of the target language in the classroom. This literature review of five research articles shows that it is beneficial for pupils’ language development to have lessons where mainly the target language is used. It is for example beneficial for pupils’ ability to speak, their pronunciation, vocabulary and ability to use language strategies. The results show that there are challenges as well, especially for the teachers, since use of the target language presumes that the teacher has good language skills and is capable of scaffolding each pupil at their individual level and in their zone of proximal development. Furthermore, there are challenges like differences in pupils’ skill level, creating tasks that both motivate and stimulate, and creating a safe learning environment. Even though the results in this thesis are limited, it is still obvious that it is an important area, where more research is necessary in order to assist teachers in how to teach English as effectively as possible.

Effects of approach velocity and foot-target characteristics on the visual regulation of step length

Two questions emerge from the literature concerning the perceptual-motor processes underlying the visual regulation of step length. The first concerns the effects of velocity on the onset of visual control (VCO), when visual regulation of step length begins during goal-directed locomotion. The second concerns the effects of different obstacles such as a target or raised surface on step length regulation. In two separate experiments, participants (Experiment 1 & 2: n=12, 6 female, 6 male) walked, jogged, or sprinted towards an obstacle along a 10 m walkway, consisting of two marker-strips with alternating black and white 0.50 m markings. Each experiment consisted of three targeting or obstacle tasks with the requirement to both negotiate and continue moving (run-through) through the target. Five trials were conducted for each task and approach speed, with trials block randomised between the six participants of each gender. One 50 Hz video camera panned and filmed each trial from an elevated position, adjacent to the walkway. Video footage was digitized to deduce the gait characteristics. Results for the targeting tasks indicate a linear relationship between approach velocity and accuracy of final foot placement (r=0.89). When foot placement was highly constrained by the obstacle step length shortened during the entire approach. VCO was found to occur at an earlier tau-margin for lower approach velocities for both experiments, indicating that the optical variable ‘tau' is affected by approach velocity. A three-phase kinematic profile was found for all tasks, except for the take-off board condition when sprinting. Further research is needed to determine whether this velocity affect on VCO is due to ‘whole-body' approach velocity or whether it is a function of the differences between gait modes.

Blaming the target of sexual harrassment: impact of gender role, sexist attitudes and work role.

This study was conducted to examine factors associated with blaming the target of sexual harassment. Participants' experiences of sexual harassment, sexist attitudes, gender, gender role identity, age, worker or student status, and belief in a just world were included as independent variables. Level of blame was evaluated using a series of 12 vignettes that manipulated the gender of the target and harasser as well as the seriousness of the harassing behavior. The sample comprised 30 female and 32 male workers from two workplaces, whose ages ranged from 18 to 65 (M = 35) years, and 102 female and 18 male university students whose ages ranged from 17 to 40 (M = 21) years. Approximately 70% of the sample were from Anglo Australian background, and 30% from European, Middle Eastern or Asian background. Females experienced more sexual harassment than males did, although the male rate was higher than expected. Although the majority of subjects attributed little blame to the target, males blamed the target of sexual harassment more than females did, and workers blamed the target of harassment more than university students did. Worker status, sexist attitudes, and gender significantly predicted blame for the total sample. Gender-typing increased the blame of the target by males but not by females. Attribution of blame was significantly influenced by worker versus student status, which supports the social psychological perspective that gender-related behavior is context dependent. The findings from this study suggest that organisational culture and environment influence respondents' attitudes to sexually harassing behavior.

The effects of target length on the visual control of step length for hard and soft impacts

Adjustments to gait were examined when positioning the foot within a narrow target at the end of an approach for two impact conditions, hard and soft. Participants (6 M, 6 F) ran toward a target of three lengths along a 10-m walkway consisting of two marker strips with alternating black and white 0.5-m markings. Five trials were conducted for each target length and impact task, with trials block randomized between the 6 participants of each gender. A 50-Hz digital video camera panned and filmed each trial from an elevated position adjacent to the walkway. Video footage was digitized to deduce the gait characteristics. A linear speed/accuracy tradeoff between target length and approach time was found for both impact tasks (hard, r = 0.99, p < 0.01; soft, r = 0.96, p < 0.05). For the hard-impact task, visual control time increased linearly (r = 0.99, p < 0.05) when whole-body approach velocity decreased. Visual control time was unaffected by whole-body approach velocity in the soft-impact task. A constant tau-margin of 1.08 describes the onset of visual control when approaching a target while running, with the control of braking during visual control described by a tau-dot of –0.85. Further research is needed to examine the control of braking in different targeting tasks.