Monitoring UV-induced signalling pathways in an ex vivo skin organ culture model using phospho-antibody array.

We investigated UV-induced signalling in an ex vivo skin organ culture model using phospho-antibody array. Phosphorylation modulations were analysed in time-course experiments following exposure to solar-simulated UV and validated by Western blot analyses. We found that UV induced P-p38 and its substrates, P-ERK1/2 and P-AKT, which were previously shown to be upregulated by UV in cultured keratinocytes and in vivo human skin. This indicates that phospho-antibody array applied to ex vivo skin organ culture is a relevant experimental system to investigate signalling events following perturbations. As the identified proteins are components of pathways implicated in skin tumorigenesis, UV-exposed skin organ culture model could be used to investigate the effect on these pathways of NMSC cancer drug candidates. In addition, we found that phospho-HCK is induced upon UV exposure, producing a new candidate for future studies investigating its role in the skin response to UV and UV-induced carcinogenesis.

Organophosphorus flame retardants in mangrove sediments from the Pearl River Estuary, South China.

Forty-eight surface sediments were collected from three mangrove wetlands in the Pearl River Estuary (PRE) of South China to investigate the distribution of organophosphorus flame retardants (OPFRs) and the relationship between OPFRs and microbial community structure determined by phospholipid fatty acid. Concentrations of ΣOPFRs in mangrove sediments of the PRE ranged from 13.2 to 377.1 ng g-1 dry weight. Levels of ΣOPFRs in mangrove sediments from Shenzhen and Guangzhou were significantly higher than those from Zhuhai, indicating that OPFRs were linked to industrialization and urbanization. Tris(chloropropyl)phosphate was the predominant profile of OPFRs in mangrove sediments from Shenzhen (38.9%) and Guangzhou (35.0%), while the composition profile of OPFRs in mangrove sediments from Zhuhai was dominated by tris(2-chloroethyl) phosphate (25.5%). The mass inventories of OPFRs in the mangrove sediments of Guangzhou, Zhuhai and Shenzhen were 439.5, 133.5 and 662.3 ng cm-2, respectively. Redundancy analysis revealed that OPFRs induced a shift in the structure of mangrove sediment microbial community and the variations were significantly correlated with tris(1,3-dichloro-2-propyl)phosphate and tris(2-butoxyethyl) phosphate.

Determinación de anticuerpos anti-proteínas citrulinadas / No disponible

No disponible

Características clínicas, evolución y pronóstico de la nefropatía membranosa idiopática en función de la presencia de anticuerpos contra el receptor tipo M de la fosfolipasa A2 / Clinical features, course and prognosis of idiopathic membranous nephropathy depending on the presence of antibodies against M-type phospholipase A2 receptor

En la nefropatía membranosa (NM), la presencia de anticuerpos antirreceptor tipo M de fosfolipasa A2 se considera altamente específica para las formas idiopáticas, pero no se ha demostrado que la presencia de dichos anticuerpos se asocie a un determinado perfil clínico. Objetivo: Analizar si existe alguna diferencia en cuanto al perfil clínico inicial, evolución y pronóstico entre pacientes con NM idiopática en función de la presencia de anticuerpos anti-PLA2R. Métodos: Se estudió a 85 enfermos conNMidiopática, 55 eran anti-PLA2R positivos y 30 negativos. Se registraron las variables clínicas, bioquímicas y anatomopatológicas al momento del diagnóstico, la frecuencia de remisión espontánea, la incidencia de respuesta al tratamiento de primera línea, la frecuencia y número de recidivas, la supervivencia de la función renal libre de tratamiento sustitutivo renal, la supervivencia de la función renal libre de insuficiencia renal crónica y la frecuencia de aparición de enfermedades neoplásicas, infecciosas o autoinmunes durante el seguimiento. Resultados: Al momento del diagnóstico, los enfermos anti-PLA2R negativos presentaron significativamente mayor edad y frecuencia de remisión espontánea. No se apreciaron diferencias en la respuesta al tratamiento de primera línea, frecuencia ni número de recidivas, supervivencia de la función renal libre de tratamiento sustitutivo renal ni supervivencia de función renal libre de insuficiencia renal crónica. Conclusiones: Los enfermos conNMidiopática anti-PLA2R negativos presentaronmayor edad, menor filtrado glomerular inicial y mayor frecuencia de remisión espontánea que los enfermos anti-PLA2R positivos. Sin embargo, entre ambos grupos de enfermos, no se observaron diferencias en cuanto a la respuesta y al tratamiento, aparición de recidivas ni pronóstico final (AU)

In membranous nephropathy, the presence of antibodies against M-type phospholipase A2 receptor is considered highly specific for idiopathic forms. However, no specific association to a particular clinical profile has been found for such antibodies. Objective: To assess potential differences in initial clinical profile, course and prognosis of idiopathic membranous nephropathy depending on the presence of anti-PLA2R antibodies. Methods: Eighty-five patients with idiopathic membranous nephropathy were included (55 anti-PLA2R-positive and 30 anti-PLA2R-negative). Clinical, biochemical and pathological variables were recorded at the time of diagnosis. Frequency of spontaneous remission, incidence of response to first-line therapy, frequency and number of recurrences, survival of renal function free from renal replacement therapy, survival of renal function free from chronic renal insufficiency and frequency of occurrence of malignant, infectious or autoimmune diseases during follow-up were recorded. Results: At the time of diagnosis, anti-PLA2R-negative patients were significantly older and had a higher frequency of spontaneous remission. No differences were noted in the response to first-line treatment, frequency and number of recurrences, survival of renal function free from renal replacement therapy, or survival of renal function free from chronic renal insufficiency. Conclusions: Anti-PLA2R-negative patients with idiopathic membranous nephropathy were older and experienced spontaneous remission more often than anti-PLA2R-positive patients. No differences in terms of treatment response, recurrences, and final prognosis were observed between both groups of patients (AU)

Factores de transferencia en la terapéutica médica / Transfer factors in medical therapy

Los factores de transferencia (FT) son mensajeros peptídicos producidos por linfocitos T activados como parte de la inmunidad celular que actúa en linfocitos vírgenes a través de FT inductores, supresores y específicos de antígeno. Los FT no son inmunógenos porque no son específicos de especie, ya que contienen una secuencia consenso de aminoácidos LLYAQDL/VEDN. De igual manera, el FT extraído de leucocitos de los humanos puede transferir inmunidad de una a otra especie. Los extractos del FT son complejos y contienen más de 200 moléculas con pesos moleculares de 1–20kDa. Los factores de transferencia específicos de antígeno (FTE) tienen pesos moleculares entre 3,5–5kDa. El FT es fácil de preparar, es bien tolerado y no contiene antígenos HL-A contra los que el receptor pueda reaccionar; además, pueden utilizarse como terapia coadyuvante en diversas enfermedades (AU)

A novel cell-based, high-content assay for phosphorylation of Lats2 by Aurora A.

Aurora A kinase is a key regulator of mitosis, which is upregulated in several human cancers, making it a potential target for anticancer therapeutics. Consequently, robust medium- to high-throughput cell-based assays to measure Aurora A kinase activity are critical for the development of small-molecule inhibitors. Here the authors compare measurement of the phosphorylation of two Aurora A substrates previously used in high-content screening Aurora A assays, Aurora A itself and TACC3, with a novel substrate Lats2. Using antibodies directed against phosphorylated forms of Aurora A (pThr288), P-TACC3 (pSer558), and P-Lats2 (pSer83), the authors investigate their suitability in parallel for development of a cell-based assay using several reference Aurora inhibitors: MLN8054, VX680, and AZD1152-HQPA. They validate a combined assay of target-specific phosphorylation of Lats2 at the centrosome and an increase in mitotic index as a measure of Aurora A activity. The assay is both sensitive and robust and has acceptable assay performance for high-throughput screening or potency estimation from concentration-response assays. It has the advantage that it can be carried out using a commercially available monoclonal antibody against phospho-Lats2 and the widely available Cellomics ArrayScan HCS reader and thus represents a significant addition to the tools available for the identification of Aurora A specific inhibitors.

Cytometry of intracellular signaling: from laboratory bench to clinical application.

The analysis of signaling pathways based on combinations of phospho-specific antibodies is now a well-recognized flow cytometry technique. Despite its wide-ranging potential in the fields of biology, industry, and medicine, it has been relatively slow to gain widespread use, and is often considered to be technically challenging. In this chapter, we detail protocols developed in our laboratory for monitoring signaling pathways in blood samples based on combinations of phospho-specific antibodies. Emphasis is placed on clinical application. The assays have a modular design, with a core protocol for whole blood fixation and lysis, a suite of agents that can acutely activate or inhibit the different signaling pathways, and a wide range of phospho-specific antibodies as the readout.

Regulation of transcription of the Aedes albopictus cecropin A1 gene: A role for p38 mitogen-activated protein kinase.

Regulation of the Aedes albopictus cecropin A1 promoter was studied to provide insight into the transcriptional control of this antimicrobial peptide (AMP) gene in mosquitoes. Gene expression levels of cecropin A1 increased in A. albopictus C6/36 cells in response to heat-killed Escherichiacoli. Reporter gene assays incorporating -757 to +32 of the A. albopictus cecropin A1 promoter revealed that E. coli could induce expression in these cells with more pronounced expression than that seen with lipopolysaccharide (LPS). Analysis of deletion constructs demonstrated that the 5' boundary of the regulatory region for the activation of this AMP was located between -173 and -64. Western blotting with anti-phospho-specific antibodies demonstrated that p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK) were activated by LPS, whereas only p38 MAPK was activated by E. coli. Moreover, pharmacological experiments revealed that pre-incubation of cells with the p38 MAPK inhibitor SB203580 resulted in a striking activation of the cecropin A1 promoter following immune challenge, demonstrating that p38 MAPK negatively regulates cecropin A1 promoter activity. Finally the region required for the negative regulation by p38 MAPK was identified as being between -173 and -64. This report is the first to show involvement of the p38 MAPK pathway in the negative regulation of AMP production in a mosquito.

Overview of the generation, validation, and application of phosphosite-specific antibodies.

Protein phosphorylation is a universal key posttranslational modification that affects the activity and other properties of intracellular proteins. Phosphosite-specific antibodies can be produced as polyclonals or monoclonals in different animal species, and each approach offers its own benefits and disadvantages. The validation of phosphosite-specific antibodies requires multiple techniques and tactics to demonstrate their specificity. These antibodies can be used in arrays, flow cytometry, and imaging platforms. The specificity of phosphosite-specific antibodies is key for their use in proteomics and profiling of disease.

Selection and validation of antibodies for signal transduction immunohistochemistry.

The in situ expression levels and subcellular localization of molecules involved in signal transduction using specific antibodies can be useful for prognosis and diagnosis of human diseases such as cancer. In addition, it has the potential to be helpful in monitoring biologic response to targeted therapies. The increasing availability of such antibodies makes these studies feasible. However, compared to typical immunohistochemical stains in which stabile molecules such as cytokeratins are targeted, additional -validation may be required for signal transduction immunohistochemistry.

Flow cytometric analysis of cell signaling proteins.

In recent years, techniques that combine the use of phospho-specific antibodies and multiparameter flow cytometry have been developed for the detection of protein phosphorylation at the single cell level. Flow cytometry is uniquely suited for this type of analysis, as it can measure functional and phenotypic markers in the context of complex cell populations. Phosphorylation can be assessed simultaneously in multiple cell subsets, and due to the small sample sizes required, and the rapid analyses of large numbers of cells in this approach, rare cell analysis is possible without the ex vivo expansion of cells.In this chapter, we detail flow cytometric protocols for the detection of intracellular phospho-proteins in samples derived from whole blood and peripheral blood mononuclear cell preparations. These protocols define steps for cell activation, fixation, permeabilization, and staining by phospho-specific and phenotyping antibodies. We discuss technical difficulties inherent to this technique and suggest solutions to commonly encountered problems. Additionally, we show examples of phospho-protein detection in lymphocyte subsets, dendritic cells, and monocytes activated with various stimuli, including mitogens, cytokines, and superantigens. Finally, we highlight a potential clinical trial application for this flow cytometric assay as a platform for pharmacodynamic monitoring of kinase inhibitors.

Signaling events initiated by kappa opioid receptor activation: quantification and immunocolocalization using phospho-selective KOR, p38 MAPK, and K(IR) 3.1 antibodies.

Psychiatric disorders including anxiety, depression, and addiction are both precipitated and exacerbated by severe or chronic stress exposure. While acutely, stress responses are adaptive, repeated exposure to stress can dysregulate the brain in such a way as to predispose the organism to both physiological and mental illness. Understanding the neuronal chemicals, cell types, and circuits involved in both normal and pathological stress responses are essential in developing new therapeutics for psychiatric diseases. Varying degrees of stressor exposure cause the release of a constellation of chemicals, including neuropeptides such as dynorphin. Neuropeptidergic release can be very difficult to directly measure with adequate spatial and temporal resolution. Moreover, the downstream consequences following release and receptor binding are numerous and also difficult to measure with cellular resolution. Following repeated stressor exposure, dynorphin is released, binds to the kappa opioid receptor (KOR), and causes activation of KOR. Agonist-activated KOR becomes a substrate for G protein receptor kinase (GRK), which phosphorylates the Ser369 residue at the C-terminal tail of the receptor in the first step in the ß-Arrestin-dependent desensitization cascade. Through the use of phospho--selective antibodies developed and validated in the laboratory, we have the tools, to assess with fine cellular resolution, the strength of behavioral stimulus required for release, time course of the release, and regional location of release. We have gone on to show that following KOR activation, both ERK 1/2 and p38 MAP kinase phosphorylation are increased through use of commercially available phospho-selective antibodies. Finally, we have identified that one effector of KOR/p38MAP kinase is K(IR) 3.1 and have developed a phospho-selective antibody against the Y12 motif of this channel. Much like KOR and p38 MAP kinase, phosphorylation of this potassium channel increases following repeated stress. The following chapter discusses immunohistochemical and quantification methods used for phospho-selective antibodies used in various brain regions following behavioral manipulations.

Direct selection of monoclonal phosphospecific antibodies without prior phosphoamino acid mapping.

In the current post-genomic era, large scale efforts are underway to functionally explore the proteome by assembling large antibody libraries. However, because many proteins are modified post-translationally to regulate their function, collections of modification-specific sensors are also needed. Here we applied a novel approach to select monoclonal phosphospecific antibodies directly from the full-length protein and without up-front phosphoamino acid identification. We chose as antigen GRASP65, a well studied Golgi phosphoprotein. Bacterially produced full-length protein was first incubated with mitotic cytosol, thus allowing modification by naturally occurring kinases, and then used directly for affinity-based antibody selection using a single chain variable fragment phagemid library. In less than 1 week, three distinct and highly functional monoclonal phosphospecific antibodies against two GRASP65 epitopes were obtained and subsequently characterized. The presented approach is carried out fully in vitro, requires no prior knowledge of the phosphoamino acid identity, and is fast and inexpensive. It therefore has great potential to be an attractive alternative to classic animal-based protocols for the selection of post-translation modification sensors and thus to become an invaluable tool in our quest to understand the proteome in all its complexity.

Direct on-membrane peptide mass fingerprinting with MALDI-MS of tyrosine-phosphorylated proteins detected by immunostaining.

We have identified tyrosine-phosphorylated proteins on membrane from A-431 human epidermoid carcinoma cells by using detection with anti-phosphotyrosine antibody followed by PMF analysis. In there, on-membrane digestion for these protein spots was carried out on microscale region using chemical inkjet technology and the resulting tryptic digests were directly analyzed by MALDI-TOF MS. Proteins identified by a database search included phosphoproteins that are known to be markedly phosphorylated on tyrosine sites after the cells are treated with epidermal growth factor (EGF). This procedure is a rapid and easily handled approach that enables both detection and identification of phosphoproteins on a single blot membrane.

Detection of phosphorylation by immunological techniques.

Phosphorylation of unlabeled proteins can be detected using immunological or enzymatic techniques. Anti-phosphotyrosine antibodies are used with immunoblots to detect tyrosine phosphorylation. This unit presents a protocol employing anti-phosphotyrosine antibodies with detection by either (125)I-labeled protein A or enhanced chemiluminescence (ECL).