T cell specific adaptor protein (TSAd) promotes interaction of Nck with Lck and SLP-76 in T cells

Background: The Lck and Src binding adaptor protein TSAd (T cell specific adaptor) regulates actin polymerization in T cells and endothelial cells. The molecular details as to how TSAd regulates this process remain to be elucidated. Results: To identify novel interaction partners for TSAd, we used a scoring matrix-assisted ligand algorithm (SMALI), and found that the Src homology 2 (SH2) domain of the actin regulator Non-catalytic region of tyrosine kinase adaptor protein (Nck) potentially binds to TSAd phosphorylated on Tyr280 (pTyr280) and pTyr305. These predictions were confirmed by peptide array analysis, showing direct binding of recombinant Nck SH2 to both pTyr280 and pTyr305 on TSAd. In addition, the SH3 domains of Nck interacted with the proline rich region (PRR) of TSAd. Pull-down and immunoprecipitation experiments further confirmed the Nck-TSAd interactions through Nck SH2 and SH3 domains. In line with this Nck and TSAd co-localized in Jurkat cells as assessed by confocal microscopy and imaging flow cytometry. Co-immunoprecipitation experiments in Jurkat TAg cells lacking TSAd revealed that TSAd promotes interaction of Nck with Lck and SLP-76, but not Vav1. TSAd expressing Jurkat cells contained more polymerized actin, an effect dependent on TSAd exon 7, which includes interactions sites for both Nck and Lck. Conclusions: TSAd binds to and co-localizes with Nck. Expression of TSAd increases both Nck-Lck and Nck-SLP-76 interaction in T cells. Recruitment of Lck and SLP-76 to Nck by TSAd could be one mechanism by which TSAd promotes actin polymerization in activated T cells. © 2015 Hem et al

Modulation of cough response by sensory inputs from the nose - role of trigeminal TRPA1 versus TRPM8 channels

BACKGROUND: Cough, the most important airways defensive mechanism is modulated by many afferent inputs either from respiratory tussigenic areas, but also by afferent drive from other organs. In animal models, modulation of cough by nasal afferent inputs can either facilitate or inhibit the cough response, depending on the type of trigeminal afferents stimulated. METHODS: In this study we addressed the question of possible bidirectional modulation of cough response in human healthy volunteers by nasal challenges with TRPA1 and TRPM8 agonists respectively. After nasal challenges with isocyanate (AITC), cinnamaldehyde, (−) menthol and (+) menthol (all 10(-3) M) nasal symptom score, cough threshold (C2), urge to cough (Cu) and cumulative cough response were measured). RESULTS: Nasal challenges with TRPA1 relevant agonists induced considerable nasal symptoms, significantly enhanced urge to cough (p<0.05) but no statistically significant modulation of the C2 and cumulative cough response. In contrast, both TRPM8 agonists administered to the nose significantly modulated all parameters including C2 (p<0.05), Cu (p<0.01) and cumulative cough response (p <0.01) documenting strong anti irritating potential of menthol isomers. CONCLUSIONS: In addition to trigeminal afferents expressing TRP channels, olfactory nerve endings, trigemino – olfactoric relationships, the smell perception process and other supramedullar influences should be considered as potential modulators of the cough response in humans

РАЗРАБОТКА КОНЦЕПЦИИ ПОСТРОЕНИЯ АППАРАТНО-ПРОГРАММНОГО КОМПЛЕКСА МОДУЛЬНОЙ КОНСТРУКЦИИ ДЛЯ ОПРЕДЕЛЕНИЯ ХАРАКТЕРИСТИК АНТЕННЫХ СИСТЕМ ПО ИЗМЕРЕНИЯМ В БЛИЖНЕЙ ЗОНЕ

Measuring the amplitude-phase distribution of the radiation field of complex antenna systems on a certain surface close to the radiating aperture allows solving the problem of reconstructing the free-space diagram in the far field and also helps in determining the influence of various structural elements and defects of radiating surfaces on formation of directional diagram. The purpose of this work was to develop a universal hardware-software complex of a modular design aimed for determining the characteristics of wide range of antenna systems in respect of measurements of the amplitude-phase distribution of the radiation field in the near zone.The equations that connect the structure of radiation fields of the antenna system at various distances from it in planar, cylindrical and spherical coordinate systems as well as structural diagrams of the hardware part of measuring complexes have been analyzed.As a result, the concept of constructing a universal hardware-software complex for measuring the radiation field of various types of antenna systems with any type of measurement surface for solving a wide range of applied problems has been developed. A modular structure of hardware and software has been proposed; it allows reconfiguring the complex rapidly in order to measure the characteristics of any particular antenna system at all stages of product development and testing, and also makes the complex economically accessible even for small enterprises and organizations.Измерения амплитудно-фазового распределения поля излучения сложных антенных систем на некоторой поверхности вблизи излучающей апертуры позволяют решить задачи восстановления пространственной диаграммы направленности антенных систем в дальней зоне и определить влияние различных конструктивных элементов и дефектов излучающих поверхностей на формирование диаграммы направленности. Целью данной работы явилась разработка универсального аппаратно-программного комплекса модульной конструкции для определения характеристик широкого круга антенных систем по измерениям амплитудно-фазового распределения поля излучения в ближней зоне. Проанализированы описанные в литературе уравнения, связывающие структуру полей излучения антенной системы на различных расстояниях от нее в планарной, цилиндрической и сферической системах координат, а также структурные схемы аппаратной части измерительных комплексов,В результате разработана концепция построения универсального аппаратно-программного комплекса для измерения поля излучения различных типов антенных систем с любым типом поверхности измерения для решения широкого круга прикладных задач. Предложена модульная структура аппаратной части комплекса и программного обеспечения, что позволяет оперативно переконфигурировать комплекс для измерения характеристик любой конкретной антенной системы на всех стадиях разработки и испытаний изделия, а также делает комплекс экономически доступным даже для небольших предприятий и организаций

ПРИМЕНЕНИЕ СТАТИСТИЧЕСКИХ МЕТОДОВ ДЛЯ ОЦЕНКИ МЕТРОЛОГИЧЕСКИХ ХАРАКТЕРИСТИК РАДИОГОЛОГРАФИЧЕСКИХ ИЗМЕРИТЕЛЬНЫХ КОМПЛЕКСОВ

Practical application of the radio-holographic method for measuring the characteristics of antennas, especially when conducting acceptance testing of systems, requires an adequate assessment of the errors in the recovery of long-range characteristics. These errors appear to be a superposition composed of various sources, having different nature, different time characteristics and different degrees of influence on the final result. The purpose of this work was the development of a practical technique for determining the influence of random errors in measuring the amplitude-phase distribution of the field of the antenna required for the accuracy of restoring long-range characteristics (primarily the antenna pattern) of the antenna, the proposed technique being based only on processing the experimental results obtained with this measuring complex.A practical method for determining the influence of random errors in measuring the amplitude-phase distribution of the field of the antenna under study on the accuracy of restoring its long-range characteristics (primarily the directional pattern) on the basis of correlation and spectral analysis has been developed and experimentally confirmed. The main advantage of the developed method in comparison with the use of mathematical modeling is that the estimation of the accuracy of the reconstruction of the directivity diagrams is based on the results of processing experimental data obtained on a specific measuring complex and does not a priori impose any preliminary requirements on the statistical parameters of errors. The developed procedure for estimating the influence of random errors can be used to develop a methodology for metrological certification of measuring systems as measuring instruments.Практическое применение радиоголографического метода измерения характеристик антенн, в особенности при проведении приемочных испытаний систем, требует адекватной оценки погрешностей восстановления дальнезонных характеристик. Указанные погрешности являются суперпозицией слагаемых от различных источников, имеющих различную природу, различные временные характеристики и различные степени влияния на конечный результат. Целью данной работы являлась разработка практической методики определения влияния случайных погрешностей измерения амплитудно-фазового распределения поля иccледуемой антенны на точность восстановления дальнезонных характеристик (в первую очередь диаграммы направленности) антенны, причем предлагаемая методика основана только на обработке экспериментальных результатов, полученных на данном измерительном комплексе.Разработана и экспериментально подтверждена практическая методика определения влияния случайных погрешностей измерения амплитудно-фазового распределения поля исследуемой антенны на точность восстановления ее дальнезонных характеристик (в первую очередь диаграммы направленности) на основе корреляционного и спектрального анализа. Основным преимуществом разработанной методики по сравнению с использованием математического моделирования является то, что оценка точности восстановления диаграмм направленности проводится по результатам обработки экспериментальных данных, полученных на конкретном измерительном комплексе, и не накладывает априори никаких предварительных требований на статистические параметры погрешностей. Разработанная процедура оценки влияния случайных погрешностей может использоваться для разработки методики метрологической аттестации измерительных комплексов как средств измерения

ПРАКТИЧЕСКАЯ РЕАЛИЗАЦИЯ АППАРАТНО-ПРОГРАММНОГО КОМПЛЕКСА ДЛЯ ПЛАНАРНЫХ ИЗМЕРЕНИЙ ХАРАКТЕРИСТИК АНТЕНН В БЛИЖНЕЙ ЗОНЕ

Directional patterns measurement of antennas is carried out by the methods of far and near zone. Measurements in the far zone are straightforward; however they have a number of disadvantages. Near zone measurement methods are free from measurement shortcomings in the far zone, but it requires complex and expensive equipment for its implementation.Earlier, the authors have developed a concept of hardware and software modular design complex to determine antenna system characteristics as per measurements in the near zone. This concept assumes creation of a universal measuring complex to investigate various types of antenna systems with any type of measurement surface (plane, cylinder, sphere) in order to solve a wide range of applied problems. The purpose of this work lies in practical implementation of a variant of this measuring complex for measurement along the plane and determination of its metrological (hardware and software) capabilities.A working experimental sample of hardware and software complex for measuring the characteristics of antenna systems that realizes a radio-holographic method for measuring along a plane has been developed, created and practically tested. A preliminary estimation of errors in amplitude and phase measurements in the dynamic range of 45 dB and a comparison of the characteristics of several types of antennas measured in far and near zones have been made. Algorithms have been developed, a software for processing, storing and graphical display of measurement results has been created.Измерение диаграмм направленности антенн проводится методами дальней и ближней зоны. Измерения в дальней зоне являются прямыми, однако обладают рядом недостатков. Ближнезонные способы измерения свободны от недостатков измерений в дальней зоне, но требует для своей реализации сложной и дорогостоящей аппаратуры и оборудования.Ранее авторами разработана концепция построения аппаратно-программного комплекса модульной конструкции для определения характеристик антенных систем по измерениям в ближней зоне. Концепция предполагает создание универсального измерительного комплекса для исследования различных типов антенных систем с любым типом поверхности измерения (плоскость, цилиндр, сфера) для решения широкого круга прикладных задач. Целью данной работы явилась практическая реализация варианта измерительного комплекса для измерения по плоскости и определение его метрологических (аппаратных и программных) возможностей.Разработан, создан и практически опробован действующий экспериментальный образец аппаратно-программного комплекса для измерения характеристик антенных систем, реализующий радиоголографический метод измерения по плоскости. Проведена предварительная оценка погрешностей измерения амплитуды и фазы в динамическом диапазоне 45 дБ и сравнение характеристик нескольких типов антенн, измеренных в дальней и ближней зонах. Разработаны алгоритмы, создано программное обеспечение по обработке, хранению и графическому отображению результатов измерения

Cbl Enforces Vav1 Dependence and a Restricted Pathway of T Cell Development

Extensive studies of pre-TCR- and TCR-dependent signaling have led to characterization of a pathway deemed essential for efficient T cell development, and comprised of a cascade of sequential events involving phosphorylation of Lck and ZAP-70, followed by phosphorylation of LAT and SLP-76, and subsequent additional downstream events. Of interest, however, reports from our lab as well as others have indicated that the requirements for ZAP-70, LAT, and SLP-76 are partially reversed by inactivation of c-Cbl (Cbl), an E3 ubiquitin ligase that targets multiple molecules for ubiquitination and degradation. Analysis of signaling events in these Cbl knockout models, including the recently reported analysis of SLP-76 transgenes defective in interaction with Vav1, suggested that activation of Vav1 might be a critical event in alternative pathways of T cell development. To extend the analysis of signaling requirements for thymic development, we have therefore assessed the effect of Cbl inactivation on the T cell developmental defects that occur in Vav1-deficient mice. The defects in Vav1-deficient thymic development, including a marked defect in DN3-DN4 transition, were completely reversed by Cbl inactivation, accompanied by enhanced phosphorylation of PLC-γ1 and ERKs in response to pre-TCR/TCR cross-linking of Vav1-/-Cbl-/- DP thymocytes. Taken together, these results suggest a substantially modified paradigm for pre-TCR/TCR signaling and T cell development. The observed consensus pathways of T cell development, including requirements for ZAP-70, LAT, SLP-76, and Vav1, appear to reflect the restriction by Cbl of an otherwise much broader set of molecular pathways capable of mediating T cell development

Environmental factors shaping the distribution of common wintering waterbirds in a lake ecosystem with developed shoreline

In this study, we tested whether the spatial distribution of waterbirds is influenced by shoreline urbanization or other habitat characteristics. We conducted monthly censuses along shoreline sections of a continental lake (Lake Balaton, Hungary) to assess the abundance of 11 common species that use this lake as a feeding and staging area during migration and winter. We estimated the degree of urbanization of the same shoreline sections and also measured other habitat characteristics (water depth, extent of reed cover, biomass of zebra mussels, distances to waste dumps and to other wetlands). We applied linear models and model averaging to identify habitat variables with high relative importance for predicting bird distributions. Bird abundance and urbanization were strongly related only in one species. Other habitat variables exhibited stronger relationships with bird distribution: (1) diving ducks and coots preferred shoreline sections with high zebra mussel biomass, (2) gulls preferred sites close to waste dumps, and (3) the abundances of several species were higher on shoreline sections close to other wetlands. Our findings suggest that the distribution of waterbirds on Lake Balaton is largely independent of shoreline urbanization and influenced by food availability and connectivity between wetlands

New Insight into the Transcarbamylase Family: The Structure of Putrescine Transcarbamylase, a Key Catalyst for Fermentative Utilization of Agmatine

Transcarbamylases reversibly transfer a carbamyl group from carbamylphosphate (CP) to an amine. Although aspartate transcarbamylase and ornithine transcarbamylase (OTC) are well characterized, little was known about putrescine transcarbamylase (PTC), the enzyme that generates CP for ATP production in the fermentative catabolism of agmatine. We demonstrate that PTC (from Enterococcus faecalis), in addition to using putrescine, can utilize L-ornithine as a poor substrate. Crystal structures at 2.5 Å and 2.0 Å resolutions of PTC bound to its respective bisubstrate analog inhibitors for putrescine and ornithine use, N-(phosphonoacetyl)-putrescine and δ-N-(phosphonoacetyl)-L-ornithine, shed light on PTC preference for putrescine. Except for a highly prominent C-terminal helix that projects away and embraces an adjacent subunit, PTC closely resembles OTCs, suggesting recent divergence of the two enzymes. Since differences between the respective 230 and SMG loops of PTC and OTC appeared to account for the differential preference of these enzymes for putrescine and ornithine, we engineered the 230-loop of PTC to make it to resemble the SMG loop of OTCs, increasing the activity with ornithine and greatly decreasing the activity with putrescine. We also examined the role of the C-terminal helix that appears a constant and exclusive PTC trait. The enzyme lacking this helix remained active but the PTC trimer stability appeared decreased, since some of the enzyme eluted as monomers from a gel filtration column. In addition, truncated PTC tended to aggregate to hexamers, as shown both chromatographically and by X-ray crystallography. Therefore, the extra C-terminal helix plays a dual role: it stabilizes the PTC trimer and, by shielding helix 1 of an adjacent subunit, it prevents the supratrimeric oligomerizations of obscure significance observed with some OTCs. Guided by the structural data we identify signature traits that permit easy and unambiguous annotation of PTC sequences

Intrinsic Structural Disorder Confers Cellular Viability on Oncogenic Fusion Proteins

Chromosomal translocations, which often generate chimeric proteins by fusing segments of two distinct genes, represent the single major genetic aberration leading to cancer. We suggest that the unifying theme of these events is a high level of intrinsic structural disorder, enabling fusion proteins to evade cellular surveillance mechanisms that eliminate misfolded proteins. Predictions in 406 translocation-related human proteins show that they are significantly enriched in disorder (43.3% vs. 20.7% in all human proteins), they have fewer Pfam domains, and their translocation breakpoints tend to avoid domain splitting. The vicinity of the breakpoint is significantly more disordered than the rest of these already highly disordered fusion proteins. In the unlikely event of domain splitting in fusion it usually spares much of the domain or splits at locations where the newly exposed hydrophobic surface area approximates that of an intact domain. The mechanisms of action of fusion proteins suggest that in most cases their structural disorder is also essential to the acquired oncogenic function, enabling the long-range structural communication of remote binding and/or catalytic elements. In this respect, there are three major mechanisms that contribute to generating an oncogenic signal: (i) a phosphorylation site and a tyrosine-kinase domain are fused, and structural disorder of the intervening region enables intramolecular phosphorylation (e.g., BCR-ABL); (ii) a dimerisation domain fuses with a tyrosine kinase domain and disorder enables the two subunits within the homodimer to engage in permanent intermolecular phosphorylations (e.g., TFG-ALK); (iii) the fusion of a DNA-binding element to a transactivator domain results in an aberrant transcription factor that causes severe misregulation of transcription (e.g. EWS-ATF). Our findings also suggest novel strategies of intervention against the ensuing neoplastic transformations

The Antidiabetic Drug Ciglitazone Induces High Grade Bladder Cancer Cells Apoptosis through the Up-Regulation of TRAIL

International audienceBACKGROUND: Ciglitazone belongs to the thiazolidinediones class of antidiabetic drug family and is a high-affinity ligand for the Peroxisome Proliferator-Activated Receptor γ (PPARγ). Apart from its antidiabetic activity, this molecule shows antineoplastic effectiveness in numerous cancer cell lines. METHODOLOGY/PRINCIPAL FINDINGS: Using RT4 (derived from a well differentiated grade I papillary tumor) and T24 (derived from an undifferentiated grade III carcinoma) bladder cancer cells, we investigated the potential of ciglitazone to induce apoptotic cell death and characterized the molecular mechanisms involved. In RT4 cells, the drug induced G2/M cell cycle arrest characterized by an overexpression of p53, p21(waf1/CIP1) and p27(Kip1) in concomitance with a decrease of cyclin B1. On the contrary, in T24 cells, it triggered apoptosis via extrinsic and intrinsic pathways. Cell cycle arrest and induction of apoptosis occurred at high concentrations through PPARγ activation-independent pathways. We show that in vivo treatment of nude mice by ciglitazone inhibits high grade bladder cancer xenograft development. We identified a novel mechanism by which ciglitazone kills cancer cells. Ciglitazone up-regulated soluble and membrane-bound TRAIL and let TRAIL-resistant T24 cells to respond to TRAIL through caspase activation, death receptor signalling pathway and Bid cleavage. We provided evidence that TRAIL-induced apoptosis is partially driven by ciglitazone-mediated down-regulation of c-FLIP and survivin protein levels through a proteasome-dependent degradation mechanism. CONCLUSIONS/SIGNIFICANCE: Therefore, ciglitazone could be clinically relevant as chemopreventive or therapeutic agent for the treatment of TRAIL-refractory high grade urothelial cancers