Synthesis and interaction of bovine serum albumin with p‐hydroxybenzoic acid derivatives

Three novel p‐hydroxybenzoic acid derivatives (HSOP, HSOX, HSCP) were synthesized from p‐hydroxybenzoic acid and sulfonamides (sulfamonomethoxine sodium, sulfamethoxazole and sulfachloropyridazine sodium) and characterized by elemental analysis, HNMR and MS. Interactions between derivatives and bovine serum albumin (BSA) were studied by fluorescence quenching spectra, UV–vis absorption spectra and time‐resolved fluorescence spectra. Based on fluorescence quenching calculation and Förster's non‐radioactive energy transfer theory, the values of the binding constants, basic thermodynamic parameters and binding distances were obtained. Experimental results indicated that the three derivatives had a strong ability to quench fluorescence from BSA and that the binding reactions of the derivatives with BSA were a static quenching process. Thermodynamic parameters showed that binding reactions were spontaneous and exothermic and hydrogen bond and van der Waals force were predominant intermolecular forces between the derivatives and BSA. Synchronous fluorescence spectra suggested that HSOX and HSCP had little effect on the microenvironment and conformation of BSA in the binding reactions but the microenvironments around tyrosine residues were disturbed and polarity around tyrosine residues increased in the presence of HSOP. Copyright © 2012 John Wiley & Sons, Ltd.     

Mx1‐Cre mediated Rgs12 conditional knockout mice exhibit increased bone mass phenotype

Regulators of G‐protein Signaling (Rgs) proteins are the members of a multigene family of GTPase‐accelerating proteins (GAP) for the Galpha subunit of heterotrimeric G‐proteins. Rgs proteins play critical roles in the regulation of G protein couple receptor (GPCR) signaling in normal physiology and human diseases such as cancer, heart diseases, and inflammation. Rgs12 is the largest protein of the Rgs protein family. Some in vitro studies have demonstrated that Rgs12 plays a critical role in regulating cell differentiation and migration; however its function and mechanism in vivo is largely unknown. Here, we generated a floxed Rgs12 allele (Rgs12^flox/flox) in which the exon 2, containing both PDZ and PTB_PID domains of Rgs12, was flanked with two loxp sites. By using the inducible Mx1‐cre and Poly I:C system to specifically delete Rgs12 at postnatal 10 days in interferon‐responsive cells including monocyte and macrophage cells, we found that Rgs12 mutant mice had growth retardation with the phenotype of increased bone mass. We further found that deletion of Rgs12 reduced osteoclast numbers and had no significant effect on osteoblast formation. Thus, Rgs12^flox/flox conditional mice provide a valuable tool for in vivo analysis of Rgs12 function and mechanism through time‐ and cell‐specific deletion of Rgs12. genesis 51:201–209, 2013. © 2013 Wiley Periodicals, Inc.     

In Vivo Epigenomic Profiling of Germ Cells Reveals Germ Cell Molecular Signatures

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Highlights? Modified small-scale ChIP-seq method applicable to small number of cells ? Genome-wide maps of H3K4me3, H3K27me3, H3K27ac, and H2BK20ac of germ cells in vivo ? Identification of active and inactive regulatory elements in germ cells in vivo ? Germ cell H3K27me3 regions are enriched for retrotransposon repeats     

Quantitative Measurement of Phosphoproteome Response to Osmotic Stress in Arabidopsis Based on Library-Assisted eXtracted Ion Chromatogram (LAXIC)

Global phosphorylation changes in plants in response to environmental stress have been relatively poorly characterized to date. Here we introduce a novel mass spectrometry-based label-free quantitation method that facilitates systematic profiling plant phosphoproteome changes with high efficiency and accuracy. This method employs synthetic peptide libraries tailored specifically as internal standards for complex phosphopeptide samples and accordingly, a local normalization algorithm, LAXIC, which calculates phosphopeptide abundance normalized locally with co-eluting library peptides. Normalization was achieved in a small time frame centered to each phosphopeptide to compensate for the diverse ion suppression effect across retention time. The label-free LAXIC method was further treated with a linear regression function to accurately measure phosphoproteome responses to osmotic stress in Arabidopsis. Among 2027 unique phosphopeptides identified and 1850 quantified phosphopeptides in Arabidopsis samples, 468 regulated phosphopeptides representing 497 phosphosites have shown significant changes. Several known and novel components in the abiotic stress pathway were identified, illustrating the capability of this method to identify critical signaling events among dynamic and complex phosphorylation. Further assessment of those regulated proteins may help shed light on phosphorylation response to osmotic stress in plants.Phosphorylation plays a pivotal role in the regulation of a majority of cellular processes via signaling transduction pathways. During the last decade, quantitative phosphoproteomics has become a powerful and versatile platform to profile signaling pathways at a system-wide scale. Multiple signaling networks in different organisms have been characterized through global phosphorylation profiling (1–3), which has evolved over the years with highly optimized procedures for sample preparation and phosphopeptide enrichment, high resolution mass spectrometry, and data analysis algorithms to identify and quantify thousands of phosphorylation events (4–8).Quantitative phosphoproteomics can be achieved mainly by two major techniques, stable isotope labeling and label-free quantitation. Isotope labeling prior to liquid chromatography-mass spectrometry (LC-MS)¹ has been widely used, including metabolic labeling such as stable isotope labeling by amino acids in cell culture (SILAC), chemical labeling such as multiplexed isobaric tags for relative and absolute quantification (iTRAQ) and isotope-coded affinity tags (ICAT) (9–12). On the other hand, label-free quantitation has gained momentum in recent years (13–15), as no additional chemistry or sample preparation steps are required. Compared with stable isotope labeling, label-free quantitation has higher compatibility with the source of the samples, the number of samples for comparison, and the instrument choice.Many label-free approaches, in particular to phosphoproteomics, are based on ion intensity (16, 17), but they are relatively error-prone because of run-to-run variations in LC/MS performance (18). In theory, such systematic errors can be corrected by spiking an internal standard into every sample to be compared. Several methods based on internal standard spiking have been reported so far. Absolute quantification peptide technology (AQUA) (19), for example, uses synthetic peptides with isotope labeling for absolute quantitation. For a global quantitative proteomics study, it is unrealistic to spike-in all reference peptides. Another labeling reference method, spike-in SILAC appears to be a promising technique to quantify the proteome in vivo with multiplex capability and it can be extended to clinical samples (20). One solution to large-scale quantitation without any isotope labeling is pseudo internal standard approach (21), which selects endogenous house-keeping proteins as the internal standard so that no spike-in reagent is required. However, finding a good pseudo internal standard remains a challenge for phosphoproteome samples. Spike-in experiments are an alternative way to improve normalization profile. Some internal standard peptides such as MassPREP^TM (Waters) were already widely used. Other groups spiked an identical amount of standard protein into samples prior to protein digestion (22–24). There are two major normalization procedures. In one approach, sample peptides were normalized to the total peak intensity of spike-in peptides (25). Alternatively, the digested peptides were compared at first and the normalization factor was determined in different ways such as the median (26) or average of ratios (27). However, spiking an identical amount of standard proteins into phosphoproteomic samples before protein digestion may not be compatible with phosphoproteomic analyses which typically require a phosphopeptide enrichment step. Spectral counting has been extensively applied in large sets of proteomic samples because of its simplicity but the method is often not reliable for the quantitation of phosphoproteins, which are typically identified by single phosphopeptides with few spectra (28–30). Many software packages have been implemented to support the wide variety of those quantitation techniques, including commercial platforms such as Progenesis LC-MS^TM, Mascot Distiller^TM, PEAKS Q^TM, etc., as well as open-source software packages including MaxQuant (31), PEPPeR (32), Skyline (33), etc.In this study, we have devised a novel label-free quantitation strategy termed Library Assisted eXtracted Ion Chromatogram (LAXIC) for plant phosphoproteomic analyses with high accuracy and consistency (Fig. 1). The approach employs synthetic peptide libraries as the internal standard. These peptides were prepared to have proper properties for quality control assessments and mass spectrometric measurements. In particular, peptides were designed to elute sequentially over an entire LC gradient and to have suitable ionization efficiency and m/z values within the normally scanned mass range. Local normalization of peak intensity is performed using Loess Procedure, a data treatment adopted from cDNA microarray data analysis (34). To monitor the diverse ion suppression effect across retention time, the local normalization factors (LNFs) are determined by internal standard pairs in individual time windows. Finally, samples will be quantified with LNFs in order to correct variance of LC-MS conditions. This quantification occurs in a small time frame centered to each target peptide.Open in a separate windowFig. 1.Work flow for the LAXIC strategy to quantify the phosphorylation change in response to osmotic stress. A, Schematic representation of the LAXIC algorithm. First, all the chromatographic peaks were aligned and the ratios were calculated. Second, the normalization factors which equal to ratios of library peptide peaks between MS runs were chosen to construct normalization curve. Third, sample peptide peak ratios were normalized against predicted normalization factor corresponding to certain retention time. B, Schematic representation of quantitative phosphoproteomics. Plants either treated with mannitol or PBS were lysed and mixed proportionally at first. Following peptide digestion and enrichment, phosphopeptides were identified and further quantified through LAXIC incorporated with self-validating process using thelinear regression model to analyze the fold change (fold), linearity (R²) and accuracy (%Acc).Water deficit and salinity causes osmotic stress, which is a major environmental factor limiting plant agricultural productivity. Osmotic stress rapidly changes the metabolism, gene expression and development of plant cells by activating several signaling pathways. Several protein kinases have been characterized as key components in osmotic stress signaling. Arabidopsis histidine kinase AHK1 can complement the histidine kinase mutant yeast, which can act as the osmosensor in yeast (35). Overexpression of AHK1 gene increases the drought tolerance of transgenic plants in Arabidopsis (36). Similar to yeast, the MAPK kinase cascade is also involved in osmotic stress response in plants. It is reported that AtMPK3, AtMPK6, and tobacco SIPK can be activated by NaCl or mannitol, and play positive roles in osmotic signaling (37, 38). MKK7 and MKKK20 may act as the up-stream kinase in the kinase cascade (39). Involvement of some calcium-dependent protein kinases, such as AtCPK21, AtCPK6, and OsCPK7 (CDPK) in osmotic stress signaling has also been reported (40–42). Another kinase family, SNF1-related protein kinase (SnRK) 2, also participates in osmotic stress response. In Arabidopsis, there are ten members in the SnRK2 family. Five from the ten SnRK2s, SnRK2, 3, 6, 7, and 8, can be activated by abscisic acid (ABA) and play central roles in ABA-receptor coupled signaling (43, 44). Furthermore, all SnRK2s except SnRK2.9 can be activated by NaCl or mannitol treatment (43). The decuple mutant of SnRK2 showed a strong osmotic hypersensitive phenotype (45). It is proposed that protein kinases including MAPK and SnRK2s have a critical function in osmotic stress (46), but the detailed mechanism and downstream substrates or target signal components are not yet clarified. We applied, therefore, the LAXIC approach with a self-validating method (47) to profile the osmotic stress-dependent phosphoproteome in Arabidopsis by quantifying phosphorylation events before and after mannitol treatment. Among a total of over 2000 phosphopeptides, more than 400 of them are dependent on osmotic stress. Those phosphoproteins are present on enzymes participating in signaling networks that are involved in many processes such as signal transduction, cytoskeleton development, and apoptosis. Overall, LAXIC represents a powerful tool for label-free quantitative phosphoproteomics.     

Identification of Direct Tyrosine Kinase Substrates Based on Protein Kinase Assay-Linked Phosphoproteomics

Protein kinases are implicated in multiple diseases such as cancer, diabetes, cardiovascular diseases, and central nervous system disorders. Identification of kinase substrates is critical to dissecting signaling pathways and to understanding disease pathologies. However, methods and techniques used to identify bona fide kinase substrates have remained elusive. Here we describe a proteomic strategy suitable for identifying kinase specificity and direct substrates in high throughput. This approach includes an in vitro kinase assay-based substrate screening and an endogenous kinase dependent phosphorylation profiling. In the in vitro kinase reaction route, a pool of formerly phosphorylated proteins is directly extracted from whole cell extracts, dephosphorylated by phosphatase treatment, after which the kinase of interest is added. Quantitative proteomics identifies the rephosphorylated proteins as direct substrates in vitro. In parallel, the in vivo quantitative phosphoproteomics is performed in which cells are treated with or without the kinase inhibitor. Together, proteins phosphorylated in vitro overlapping with the kinase-dependent phosphoproteome in vivo represents the physiological direct substrates in high confidence. The protein kinase assay-linked phosphoproteomics was applied to identify 25 candidate substrates of the protein-tyrosine kinase SYK, including a number of known substrates and many novel substrates in human B cells. These shed light on possible new roles for SYK in multiple important signaling pathways. The results demonstrate that this integrated proteomic approach can provide an efficient strategy to screen direct substrates for protein tyrosine kinases.Protein phosphorylation plays a pivotal role in regulating biological events such as protein–protein interactions, signal transduction, subcellular localization, and apoptosis (1). Deregulation of kinase-substrate interactions often leads to disease states such as human malignancies, diabetes, and immune disorders (2). Although a number of kinases are being targeted to develop new drugs, our understanding of the precise relationships between protein kinases and their direct substrates is incomplete for the majority of protein kinases (3). Thus, mapping kinase–substrate relationships is essential for the understanding of biological signaling networks and the discovery and development of drugs for targeted therapies (4). Toward this goal, various in vitro kinase assays using synthetic peptide libraries (5), phage expression libraries (6), protein arrays (7–9), or cell extracts (10, 11) have been explored for the screening of kinase substrates.Besides classical biochemical and genetic methods, mass spectrometry-based high throughput approaches have become increasingly attractive because they are capable of sequencing proteins and localizing phosphorylation sites at the same time. Mass spectrometry-based proteomic methods have been extensively applied to kinase-substrate interaction mapping (12) and global phosphorylation profiling (13–15). Although thousands of phosphorylation events can be inspected simultaneously (16, 17), large-scale phosphoproteomics does not typically reveal direct relationships between protein kinases and their substrates.Recently, several mass spectrometry-based proteomic strategies have been introduced for identifying elusive kinase substrates (7, 18, 19). Taking advantage of recent advances of high speed and high-resolution mass spectrometry, these methods used purified, active kinases to phosphorylate cell extracts in vitro, followed by mass spectrometric analysis to identify phosphoproteins. These approaches commonly face the major challenge of distinguishing phosphorylation events triggered by the kinase reaction from background signals introduced by endogenous kinase activities (20). To dissect the phosphorylation cascade, Shokat and colleagues developed an approach named Analog-Sensitive Kinase Allele (ASKA)¹ (21). In their approach, a kinase is engineered to accept a bulky-ATP analog exclusively so that direct phosphorylation caused by the analog-sensitive target kinase can be differentiated from that of wild type kinases. As a result, indirect effects caused by contaminating kinases during the in vitro kinase assay are largely eliminated. ASKA has recently been coupled with quantitative proteomics, termed Quantitative Identification of Kinase Substrates (QIKS) (12), to identify substrate proteins of Mek1. Recently, one extension of the ASKA technique is for the analog ATP to carry a γ-thiophosphate group so that in vitro thiophosphorylated proteins can be isolated for mass spectrometric detection (22–24). In addition to ASKA, radioisotope labeling using [γ-³²P]ATP (10), using concentrated purified kinase (25), inactivating endogenous kinase activity by an additional heating step (11), and quantitative proteomics (26, 27) are alternative means aimed to address the same issues. All of these methods, however, have been limited to the identification of in vitro kinase substrates.To bridge the gap between in vitro phosphorylation and physiological phosphorylation events, we have recently introduced an integrated strategy termed Kinase Assay-Linked Phosphoproteomics (KALIP) (28). By combining in vitro kinase assays with in vivo phosphoproteomics, this method was demonstrated to have exceptional sensitivity for high confidence identification of direct kinase substrates. The main drawback for the KALIP approach is that the kinase reaction is performed at the peptide stage to eliminate any problems related to contamination by endogenous kinases. However, the KALIP method may not be effective for kinases that require a priming phosphorylation event (i.e. a previous phosphorylation, on substrate or kinase, has effect on following phosphorylation) (29), additional interacting surfaces (30), or a docking site on the protein (31). For example, basophilic kinases require multiple basic resides for phosphorylation and tryptic digestion will abolish these motifs, which are needed for effective kinase reactions.We address the shortcoming by introducing an alternative strategy termed Protein Kinase Assay-Linked Phosphoproteomics (proKALIP). The major difference between this method and the previous KALIP method is the utilization of protein extracts instead of digested peptides as the substrate pool. The major issue is how to reduce potential interference by endogenous kinase activities. One effective solution is to use a generic kinase inhibitor, 5′-(4-fluorosulfonylbenzoyl)adenosine (FSBA), which was widely used for covalent labeling of kinases (32, 33), kinase isolation (34), kinase activity exploration (35, 36), and more recently kinase substrate identification by Kothary and co-workers (37). However, an extra step is required to effectively remove the inhibitor before the kinase reaction, which may decrease the sensitivity. ProKALIP addresses the issue by carrying out the kinase reaction using formerly in vivo phosphorylated proteins as candidates. This step efficiently improves the sensitivity and specificity of the in vitro kinase reaction. Coupled with in vivo phosphoproteomics, proKALIP has gained a high sensitivity and provided physiologically relevant substrates with high confidence.To demonstrate the proKALIP strategy, the protein-tyrosine kinase SYK was used as our target kinase. SYK is known to play a crucial role in the adaptive immune response, particularly in B cells, by facilitating the antigen induced B-cell receptor (BCR) signaling pathways and modulating cellular responses to oxidative stress in a receptor-independent manner (38, 39). SYK also has diverse biological functions such as innate immune recognition, osteoclast maturation, cellular adhesion, platelet activation, and vascular development (38). In addition, the expression of SYK is highly correlated to tumorigenesis by promoting cell–cell adhesion and inhibiting the motility, growth, and invasiveness of certain cancer cells (40). In this study, we attempt to identify bona fide substrates of SYK in human B cells using the proKALIP approach and demonstrate the specificity and sensitivity of this strategy.     

Insecticidal activity of recombinant baculovirus co-expressing Bacillus thuringiensis crystal protein and Kunitz-type toxin isolated from the venom of bumblebee Bombus ignitus

To improve the insecticidal activity of Autographa californica nucleopolyhedrovirus (AcMNPV), using co-expression of Bacillus thuringiensis crystal protein and a Kunitz-type toxin isolated from bumblebee Bombus ignitus venom, a recombinant AcMNPV, ApPolh5-3006BiKTI, expressing Bi-KTI under the control of early promoter from Cotesia plutellae bracovirus (CpBV) was constructed. In this recombinant virus, B. thuringiensis cry1-5 crystal protein gene was introduced into the genome by the fusion of polyhedrin-cry1-5 under the control of polyhedrin gene promoter. RT-PCR analysis indicated that both Bi-KTI and polyhedrin-cry1-5 fusion protein were successfully expressed from the infected cells. In addition, SDS-PAGE revealed that polyhedrin-cry1-5 fusion protein expressed by recombinant viruses was occluded into the polyhedra. ApPolh5-3006BiKTI showed an improved insecticidal activity against larvae of Plutella xylostella and Spodoptera exigua. At low dosage rates, it was more effective against S. exigua than on P. xylostella, but more rapid insecticidal activity was shown in P. xylostella. These results strongly suggest that co-expression of Bt toxin and Kunitz-type toxins could be successfully applied to improve the insecticidal activity of baculoviruses.     

Notes on black elytron species of Pyrrhalta Joannis and the description of a new species from China (Coleoptera,Chrysomelidae, Galerucinae)

Thirteen species of Pyrrhalta Joannis, 1865 with black elytron are reviewed. A key to species, photographs of aedeagus and habitus are provided. Pyrrhalta qianana sp. n. is described from Guizhou, China. Pyrrhalta martensi Medvedev & Sprecher-Uebersax, 1999 is newly recorded from China (Tibet).     

Cyclin B1 is an efficacy-predicting biomarker for Chk1 inhibitors

Chk1 is the major mediator of cell-cycle checkpoints in response to various forms of genotoxic stress. Although it was previously speculated that checkpoint abrogation due to Chk1 inhibition may potentiate the efficacy of DNA-damaging agents through induction of mitotic catastrophe, there has not been direct evidence proving this process. Here, through both molecular marker and morphological analysis, we directly demonstrate that specific downregulation of Chk1 expression by Chk1 siRNA potentiates the cytotoxicities of topoisomerase inhibitors through the induction of premature chromosomal condensation and mitotic catastrophe. More importantly, we discovered that the cellular cyclin B1 level is the major determinant of the potentiation. We show that downregulation of cyclin B1 leads to impairment of the induction of mitotic catastrophe and correspondingly a reduction of the potentiation ability of either Chk1 siRNA or a small molecule Chk1 inhibitor. More significantly, we have extended the study by examining a panel of 10 cancer cell-lines with different tissue origins for their endogenous levels of cyclin B1 and the ability of a Chk1 inhibitor to sensitize the cells to DNA-damaging agents. The cellular levels of cyclin B1 positively correlate with the degrees of potentiation achieved. Of additional interest, we observed that the various colon cancer cell lines in general appear to express higher levels of cyclin B1 and also display higher sensitivity to Chk1 inhibitors, implying that Chk1 inhibitor may be more efficacious in treating colon cancers. In summary, we propose that cyclin B1 is a biomarker predictive of the efficacy of Chk1 inhibitors across different types of cancers. Unlike previously established efficacy-predictive biomarkers that are usually the direct targets of the therapeutic agents, cyclin B1 represents a non-drug-target biomarker that is based on the mechanism of action of the target inhibitor. This finding may be potentially very useful for the stratification of patients for Chk1 inhibitor clinical trials and hence, maximize its chance of success.     

Extraction and purification of a lectin from small black kidney bean (Phaseolus vulgaris) using a reversed micellar system

Lectin from crude extract of small black kidney bean (Phaseolus vulgaris) was successfully extracted using the reversed micellar extraction (RME). The effects of water content of organic phase (W_o), ionic strength, pH, Aerosol-OT (AOT) concentration and extraction time on the forward extraction and the pH and ionic strength in the backward extraction were studied to optimize the extraction efficiency and purification factor. Forward extraction of lectin was found to be maximum after 15 min of contact using 50 mM AOT in organic phase with W_o 27 and 10 mM citrate-phosphate buffer at pH 5.5 containing 100 mM NaCl in the aqueous phase. Lectin was backward extracted into a fresh aqueous phase using sodium-phosphate buffer (10 mM, pH 7.0) containing 500 mM KCl. The overall yield of the process was 53.28% for protein recovery and 8.2-fold for purification factor. The efficiency of the process was confirmed by gel electrophoresis analysis.