Qi-Wei-Du-Qi-Wan and its major constituents exert an anti-asthmatic effect by inhibiting mast cell degranulation.

ETHNOPHARMACOLOGICAL RELEVANCE: In Asia, Qi-Wei-Du-Qi-Wan (QWDQW) is a traditional Chinese medicine that has been used to treat chest tightness, cough, shortness of breath, night sweats, frequent urination and asthma. QWDQW is recorded in Yi Zong Yi Ren Pian (Medical Physician's Compilation), which was written by Yang Cheng Liu during the Qing Dynasty. AIM OF THE STUDY: The traditional Chinese medicine QWDQW is composed of 7 ingredients and has been used in the treatment of asthma in Asia for hundreds of years. However, the mechanism through which QWDQW affects the immune system in the treatment of asthma is not known. Therefore, this study aimed to investigate whether QWDQW alleviates asthmatic symptoms in mice with chronic asthma induced by repeated stimulation with Dermatophagoides pteronyssinus (Der p) and to explore the underlying immune modulatory mechanism. MATERIALS AND METHODS: BALB/c mice were stimulated intratracheally (i.t.) with Der p (40 µl, 2.5 µg/µl) once weekly for 6 weeks. Thirty minutes prior to Der p stimulation, the mice were treated with QWDQW (0.5 g/kg and 0.17 g/kg) orally. Three days after the last stimulation, the mice were sacrificed, and infiltration of inflammatory cells, lung histological characteristics, gene expression of lung and serum total IgE were assessed. In other experiments, RBL-2H3 cells were stimulated with DNP-IgE/DNP-BSA and then treated with QWDQW, quercetin, ß-carotene, luteolin or a mixture of the three chemicals (Mix13) for 30 min, and the effects of the drugs on RBL-2H3 cell degranulation after DNP stimulation were determined. RESULTS: QWDQW significantly reduced Der p-induced airway hyperreactivity (AHR) and decreased total serum IgE and Der p-specific IgE levels. Histopathological examination showed that QWDQW reduced inflammatory cell infiltration and sputum secretion from goblet cells in the lungs. Gene expression analysis indicated that QWDQW reduced overproduction of IL-12、IFN-γ、IL-13、IL-4、RNATES、Eotaxin and MCP-1in lung. Additionally, QWDQW and Mix13 suppressed DNP induced RBL-2H3 degranulation, and the effect was maximal when quercetin, ß-carotene and luteolin were administered together. CONCLUSION: These results indicate that QWDQW plays a role in suppressing excessive airway reaction and in specific immune modulation in a mouse model of chronic asthma and that QWDQW suppresses mast cell degranulation at defined doses of quercetin, ß-carotene and luteolin.

A proteomics analysis to evaluate cytotoxicity in NRK-52E cells caused by unmodified Nano-Fe3O4.

We synthesized unmodified Fe3O4 nanoparticles (NPs) with particles size from 10 nm to 100 nm. We cultured NRK-52E cell lines (rat, kidney) and treated with Fe3O4 NPs to investigate and evaluate the cytotoxicity of NPs for NRK-52E cells. Through global proteomics analysis using dimethyl labeling techniques and liquid phase chromatography coupled with a tandem mass spectrometer (LC-MS/MS), we characterized 435 proteins including the programmed cell death related proteins, ras-related proteins, glutathione related proteins, and the chaperone proteins such as heat shock proteins, serpin H1, protein disulfide-isomerase A4, endoplasmin, and endoplasmic reticulum resident proteins. From the statistical data of identified proteins, we believed that NPs treatment causes cell death and promotes expression of ras-related proteins. In order to avoid apoptosis, NRK-52E cell lines induce a series of protective effects such as glutathione related proteins to reduce reactive oxygen species (ROS), and chaperone proteins to recycle damaged proteins. We suggested that, in the indigenous cellular environment, Fe3O4 NPs treatment induced an antagonistic effect for cell lines to go to which avoids apoptosis.

Networks development between nicotinic chemical probes and Ca9-22 oral cancer cells by general proteomics analyses.

Tobacco includes thousands of chemicals such as nicotine, which causes numerous diseases including oral cancer. We synthesized nicotinic acid based probes by chemical modification to identify the proteins expressed by the oral cancer cell line Ca9-22 that interact with the nicotinic functional group. Proteins belonging to human oral squamous cell carcinoma were pulled down by a probe carrier based on nicotinic acid, which was reacted with 3-aminopropyltriethoxysilane to compose nicotinic acid linked 3-aminopropyltriethoxysilane exposed on the SiO2 surface. Oral cancer cell lysates were incubated with the nicotinic acid chemical probes to identify the interactions between the nicotinic group and oral cancer cell line extracted proteins. The interactions between the chemical probes and proteins identified as their targets were confirmed by consulting chemicals databases. Interestingly, chaperone proteins (e.g., heat-shock proteins and endoplasmin) that were found to interact with nicotinic acid were identified as binding partners in ribosomal and nucleosome assembly complexes.

Phthalic acid chemical probes synthesized for protein-protein interaction analysis.

Plasticizers are additives that are used to increase the flexibility of plastic during manufacturing. However, in injection molding processes, plasticizers cannot be generated with monomers because they can peel off from the plastics into the surrounding environment, water, or food, or become attached to skin. Among the various plasticizers that are used, 1,2-benzenedicarboxylic acid (phthalic acid) is a typical precursor to generate phthalates. In addition, phthalic acid is a metabolite of diethylhexyl phthalate (DEHP). According to Gene_Ontology gene/protein database, phthalates can cause genital diseases, cardiotoxicity, hepatotoxicity, nephrotoxicity, etc. In this study, a silanized linker (3-aminopropyl triethoxyslane, APTES) was deposited on silicon dioxides (SiO2) particles and phthalate chemical probes were manufactured from phthalic acid and APTES-SiO2. These probes could be used for detecting proteins that targeted phthalic acid and for protein-protein interactions. The phthalic acid chemical probes we produced were incubated with epithelioid cell lysates of normal rat kidney (NRK-52E cells) to detect the interactions between phthalic acid and NRK-52E extracted proteins. These chemical probes interacted with a number of chaperones such as protein disulfide-isomerase A6, heat shock proteins, and Serpin H1. Ingenuity Pathways Analysis (IPA) software showed that these chemical probes were a practical technique for protein-protein interaction analysis.

Reduction of Airway Hyperresponsiveness by KWLL in Dermatophagoides-pteronyssinus-Challenged Mice.

Urine therapy has been commonly practiced in ancient civilizations including those of India, China, and Greece. The traditional Chinese medicine KWLL, the precipitation of human urine, has been used in China to alleviate the symptoms of asthma for thousands of years. However, the mechanism of action by which KWLL exerts its immunotherapy is unclear. This study attempted to elucidate the pharmacology of KWLL in mice that had been challenged recurrently by Dermatophagoides pteronyssinus (Der p). BALB/c mice were orally administered KWLL (1 g/kg) before an intratracheal (i.t.) challenge of Der p. Allergic airway inflammation and remodeling were provoked by repetitive Der p (50 µ g/mice) challenges six times at 1 wk intervals. Airway hypersensitivity, histological lung characteristics, and the expression profiles of cytokines and various genes were assessed. KWLL reduced Der p-induced airway hyperresponsiveness and inhibited eosinophil infiltration by downregulating the protein expression of IL-5 in bronchoalveolar lavage fluid (BALF). It also inhibited neutrophil recruitment by downregulating IL-17A in BALF. KWLL effectively diminished inflammatory cells, goblet cell hyperplasia, and mRNA expression of IL-6 and IL-17A in the lung. The reduction by KWLL of airway inflammatory and hyperresponsiveness in allergic asthmatic mice was mediated via immunomodulation of IL-5, IL-6, and IL-17A.

Heat shock protein 90 stabilizes nucleolin to increase mRNA stability in mitosis.

Most studies on heat shock protein 90 (Hsp90) have focused on the involvement of Hsp90 in the interphase, whereas the role of this protein in the nucleus during mitosis remains largely unclear. In this study, we found that the level of the acetylated form of Hsp90 decreased dramatically during mitosis, which indicates more chaperone activity during mitosis. We thus probed proteins that interacted with Hsp90 by liquid chromatography/mass spectrometry (LC/MS) and found that nucleolin was one of those interacting proteins during mitosis. The nucleolin level decreased upon geldanamycin treatment, and Hsp90 maintained the cyclin-dependent kinase 1 (CDK1) activity to phosphorylate nucleolin at Thr-641/707. Mutation of Thr-641/707 resulted in the destabilization of nucleolin in mitosis. We globally screened the level of mitotic mRNAs and found that 229 mRNAs decreased during mitosis in the presence of geldanamycin. Furthermore, a bioinformatics tool and an RNA immunoprecipitation assay found that 16 mRNAs, including cadherin and Bcl-xl, were stabilized through the recruitment of nucleolin to the 3'-untranslated regions (3'-UTRs) of those genes. Overall, strong correlations exist between the up-regulation of Hsp90, nucleolin, and the mRNAs related to tumorigenesis of the lung. Our findings thus indicate that nucleolin stabilized by Hsp90 contributes to the lung tumorigenesis by increasing the level of many tumor-related mRNAs during mitosis.

Urinary CD14 as a potential biomarker for benign prostatic hyperplasia - discovery by combining MALDI-TOF-based biostatistics and ESI-MS/MS-based stable-isotope labeling.

PURPOSE: Quest for specific urinary biomarkers for benign prostatic hyperplasia (BPH). EXPERIMENTAL DESIGN: Proteomics studies were conducted with urines of the training set to discovering marker candidates that could differentiate BPH from normal subjects by matching results deduced from MALDI-TOF of individual samples and results deduced from nanoLC-ESI-MS/MS-based stable isotope dimethyl labeling of two pooled samples (BPH and normal). Samples were digested before analysis and such an approach takes into account the subject-to-subject variation and differential amount, as well as protein identification. Selected markers were validated by ELISA conducted on the training set and the test set as well as another set of urines collected from prostate cancer patients. RESULTS: Nine marker candidates were identified from proteomics studies; CD14, prostate-specific antigen and pancreatic α-amylase precursor were further selected for ELISA validation. Urinary CD14 is among the best match with high specificity (>81%) for both training and test sets. In addition, from the study of prostate cancer patients, CD14 also allows the distinction of BPH from cancer with high specificity (84-100%) when combined with urinary prostate-specific antigen. CONCLUSIONS AND CLINICAL RELEVANCE: Urinary CD14 is suggested to have a high specificity in the diagnosis of BPH in distinction from normal as well as cancer subjects.

Quantitative phosphoproteomics studies using stable isotope dimethyl labeling coupled with IMAC-HILIC-nanoLC-MS/MS for estrogen-induced transcriptional regulation.

17ß-Estradiol (E2) regulates transcriptional activity partly by inducing protein-kinase cascades, leading to the phosphorylation of estrogen receptors (ERs) and other functional proteins. Many of these phosphorylation events are also modulated by growth factors. To gain an insight into E2-modulated protein phosphorylation, we applied quantitative phosphoproteomics to investigate global changes in protein phosphorylation induced by E2 in MCF-7 cells. Proteomic analyses using stable isotope dimethyl labeling coupled with immobilized metal affinity chromatography-hydrophilic interaction liquid chromatography (IMAC-HILIC) fractionation and nanoLC-MS/MS identified and quantified 2857 unique phosphorylation sites in 1338 phosphoproteins from 1 mg of total cellular protein. In addition to S118 of ERα, a 30-min E2 treatment significantly altered the status of 403 phosphorylation sites, including 112 novel sites. Interestingly, the substrate motifs for ERK1/2 were largely enriched in both the up-regulated and down-regulated phosphorylation sites. An increase in the phosphorylation on either the T202 or Y204 sites of ERK1 was observed after E2 treatment, while dual phosphorylation on both sites were not detected, implying that a feedback loop to deactivate MAPK signaling was achieved during a 30-min E2 treatment. In contrast, the PKA and CKII substrate motifs were majorly enriched among the up-regulated phosphorylation sites. Western blot analysis confirmed that E2 increased the phosphorylation level of S226 within a CKII motif of HSP90ß by a factor of 2- to 3-fold without changing the total protein expression level. E2 also up-regulated phosphorylations of S255 in HSP90ß and S353 within a CKII motif of HSP90α. These results indicated that E2 may modulate gene transcription by affecting the stability, function, and activity of many regulators through a HSP90 phosphorylation-mediated chaperoning process. This study, using a quantitative, multidimensional separation phosphoproteomic approach that required a relatively low amount of cells, provides new insights into the diversity, variability, and dynamic nature of the protein phosphorylation/dephosphorylation elicited by E2.

Mapping N-terminus phosphorylation sites and quantitation by stable isotope dimethyl labeling.

We have previously coupled stable isotope dimethyl labeling with IMAC enrichment for quantifying the extent of protein phosphorylation in vivo. The enhanced a(1) signal of dimethylated peptides served as a unique mass tag for unequivocal identification of the N-terminal amino acids. In this study, we demonstrate that the a(1) ion could further assist in mapping the precise phosphorylation site near the N-terminal region and allow the determination of the exact site and level of phosphorylation in one step by stable isotope dimethyl labeling. We show that the a(1) ion signal was suppressed for dimethylated peptides with a phosphorylation site at the N-terminus Ser/Thr residue (N-p*Ser/Thr) but was still enhanced for N-terminus Tyr residue (N-p*Tyr) or internal Ser/Thr residues (-p*Ser/Thr). Based on the dominant de-phosphorylated molecular ions and b-H(3)PO(4) ions for N-p*Ser/Thr, we propose that dimethyl labeling increases the basicity of the N-terminus and accelerates the de-phosphorylation for N-p*Ser/Thr precursors, which, however, suppresses the a(1) ion enhancement due to the resulting unsaturated covalent bond on C(alpha) of the N-terminus amino acid. Using this method, we excluded three Ser/Thr phosphorylation sites in A431 cells, two of which, however, were previously reported to be phosphorylation sites; we confirmed three known phosphorylation sites in A431 cells and quantified their ratios upon EGF treatment. Notably, we identified a novel phosphorylation site on Ser43 residue at N-terminus of the tryptic peptide derived from SVH protein in pregnant rat uteri. SVH protein has not been reported or implied with any phosphorylation event, and our data show that the Ser43 of SVH is an intrinsic phosphorylation site in pregnant rat uteri and that its phosphorylation level was slightly decreased upon c-AMP treatment.

A systematic MS-based approach for identifying in vitro substrates of PKA and PKG in rat uteri.

Protein phosphorylation is an important modulator of many cellular processes, and identification of kinase substrates provides critical insights for signal transduction. However, this identification process is often difficult and many kinase substrates remain unexplored. Herein, a systematic proteomics approach solely depending on MS detection is reported for identifying substrates of PKA and PKG, which are suspected to have similar specificity determinants, in pregnant rat uteri. Instead of radioisotopes that are commonly used to couple with MS for substrate identification, this study developed an efficient in vitro kinase assay on depleted tissue homogenates to reveal substrate candidates directly by MS. To facilitate MS detection, exogenous phosphatases were added to remove intrinsic phosphorylation followed by a heating step to inactivate all enzymes. No observable interference caused by endogenous kinases or background phosphorylation was detected in the control experiment in which no kinase was externally added. A total of 61 and 12 substrate candidates were identified in vitro for PKA and PKG, respectively, and most of these identified sites contain consensus motifs of each kinase with only a few sites overlapped, indicating a good specificity. Moreover, differential phosphoproteomics analysis using stable isotope dimethyl labeling and MS was performed to detect the change of protein phosphorylation upon kinase stimulation in vivo. Four identified in vitro PKA substrates including three reported sites on HSP27 or filamin A were significantly phosphorylated in vivo, giving them high confidence as physiological substrates in pregnant rat uteri. Moreover, telokin, a known PKG substrate on S1880, and actin-binding proteins such as Arp 3, titin, and desmuslin were also identified to be in vitro PKG substrates in pregnant rat uteri. These proteins are all expected to be involved in the regulation of actin-mediated cytoskeletal remodeling.

Gold nanoparticle-assisted protein enrichment and electroelution for biological samples containing low protein concentration--a prelude of gel electrophoresis.

Protein enrichment is essential for biological samples that contain low protein concentrations, especially for proteomic studies that require sufficient quantities for subsequent MS analysis. Traditional precipitation methods, however, are limited in the sample volume and protein concentration required to cause efficient precipitations. We showed that gold nanoparticles (Au-NPs) can be easily applied to concentrate proteins from more than 15 mL of human urine, in which the total protein concentration is less than 1.4 ppm. Moreover, Au-NP-aggregated proteins can be directly applied to gel electrophoresis for Au-NP-protein dissociation followed by free protein separation as well as for the subsequent in-gel digestion and protein identification by mass spectrometry. We compared this method with trichloroacetic acid (TCA) precipitation method, one of the most common precipitation methods, and TCA method showed no enrichment effect for protein samples with large volumes (>2 mL) or with low protein concentrations (4 ppm). Therefore, Au-NP aggregation is not only a simple and efficient method for enriching a broad range of proteins, it is also particularly useful for concentrating proteins from a relatively large volume of dilute biological fluids, under which TCA method is ineffective.

Quantitation of protein phosphorylation in pregnant rat uteri using stable isotope dimethyl labeling coupled with IMAC.

Quantitative analysis of protein phosphorylation provides important insights into molecular signaling mechanisms and a better understanding of many cellular processes. In this study, we coupled stable isotope dimethyl labeling with immobilized metal affinity chromatography (IMAC) enrichment to quantify protein phosphorylation at MS-determined phosphorylation sites. The proposed method was first characterized using alpha- and beta-casein as two model phosphoproteins, and further applied to the analysis of pregnant rat uteri with and without treatment with 8-bromo-cGMP. Dimethyl labeling has several significant advantages: global, fast (within 5 min) and complete (near 100%). Our results indicate that the labeling has no adverse effect on the IMAC enrichment for tryptic peptides having single and multiple phosphorylation sites. Moreover, the enhanced a1 signal and the complete reaction by dimethyl labeling provide unequivocal identification of both the N-terminal amino acid and the number of the labeling site. Using these two criteria in data validation, which is particularly important for identifying phosphoproteins, we found that the confidence in interpreting dimethyl-labeled peptides had greatly increased. In the analysis of late gestation rat uteri, the abundance ratio between treated and un-treated phosphopeptide signals ranged from 0.51 to 1.69 with an average of around 1.01 +/- 0.25. The obtained ratio of the phosphorylation levels at Ser 15 of HSP27 was further confirmed by the consistent results obtained from Western blot analyses. Based on the analysis of the results, it is interesting to note that the activated cGMP dependent protein kinase G (PKG) seems to affect the phosphorylation of proteins associated with the inhibition of cell migration and proliferation, redistribution of actin-associated proteins, and the increase of protein synthesis in late-gestation uteri. These observations provide important evidence suggesting that activated PKG may play a critical role in the shift of pregnant uteri from proliferative to hypertrophic states.

Mycotic aneurysms and death in a hemodialysis patient.

A patient with newly diagnosed end-stage renal disease (ESRD) received a femoral catheter for hemodialysis (HD). Shortly thereafter he developed fever, and blood cultures grew methicillin-resistant Staphylococcus aureus. The catheter was removed and the patient was treated with both vancomycin and rifampin; however, blood culture positivity persisted. The cerebrospinal fluid showed sterile meningitis. Subsequent imaging studies demonstrated aortic valve endocarditis and multiple mycotic aneurysms that appeared to include the intra- and extracranial vessels. The patient eventually died from sepsis. This case illustrates the aggressive and invasive nature of systemic infection with S. aureus and underscores the high morbidity and mortality associated with infections related to HD catheters.

Photopolymerized microtips for sample preparation in proteomic analysis.

We demonstrate a novel method for the fabrication of disposable plastic microtips, which we name "EasyTip", by a photopolymerization technique. C18 reversed-phase (C18) and ion metal affinity chromatography (IMAC) beads were immobilized on a plastic pipette tip, made of polypropylene materials, by photo-initiated polymerization. The fabricated EasyTips can be manipulated using commercial pipettes for wash/elution of minute amount of biological samples (< 10 microL) and can be applied for mass spectrometry (MS)-based proteomic analysis, in which the detection sensitivity depends critically on the optimal sample preparation. The recovery of a sample of 25 fmol of tryptic hemoglobin digest loaded in a C18 EasyTip was near 100% and we estimated the loading capacity to be around 0.4-2.0 microg of total proteins or peptides, which is well above a sufficient quantity for MS analysis. The effectiveness of the C18 EasyTips in enhancing the detection sensitivity of matrix-assisted laser desorption/ionization (MALDI)-MS signal, and thus providing a greater sequence coverage, was also demonstrated by the analysis of hemoglobin digest and the in-gel digested epidermal growth factor receptor (EGFR) protein from A431 cell lysate. We also demonstrated the usefulness of the immobilized IMAC EasyTips in extracting the signal of tryptic phosphopeptides of beta-casein (10 pmol) having one and four phosphorylation sites by using an IMAC EasyTip prior to off-line analysis by MS. The combination of IMAC EasyTips and MALDI-MS allowed the unambiguous identification of phosphopeptides based on the phosphatase assay as well as the post-source decay. Compared to other miniaturized devices, this fabrication method is simple, cheap, and requires less human intervention. Moreover, the method of manipulating the EasyTips is straightforward and can be automated readily by a robotic system for high-throughput analysis.

A convenient method to extract matrix-assisted laser desorption/ionization mass spectrometry spectra from phosphate-containing peptide mixtures.

Here we report that the addition of HCl or HNO(3) to the matrix at a limited concentration dramatically increases the signal-to-noise ratio of the matrix-assisted laser desorption/ionization mass spectrometry spectrum of phosphate-containing peptide mixtures such as those obtained from an immobilised metal affinity capture eluent or a phosphate-containing tryptic digest. These improved spectra permitted both peptide identification and the determination of protein phosphorylation sites. In comparison to existing methods for removing salts, this method requires less sample manipulation and thus less sample loss is expected.