Search

Phosphorylation by serine-threonine and tyrosine kinases is critical for determining protein function. Array-based platforms for measuring reporter peptide signal levels allow for differential phosphorylation analysis between conditions for distinct active kinases. Peptide array technologies like the PamStation12 from PamGene allow for generating high-throughput, multi-dimensional, and complex functional proteomics data. As the adoption rate of such technologies increases, there is an imperative need for software tools that streamline the process of analyzing such data. We present Kinome Random Sampling Analyzer (KRSA), an R package and R Shiny web-application for analyzing kinome array data to help users better understand the patterns of functional proteomics in complex biological systems. KRSA is an All-In-One tool that reads, formats, fits models, analyzes, and visualizes PamStation12 kinome data. While the underlying algorithm has been experimentally validated in previous publications, we demonstrate KRSA workflow on dorsolateral prefrontal cortex (DLPFC) in male (n = 3) and female (n = 3) subjects to identify differential phosphorylation signatures and upstream kinase activity. Kinase activity differences between males and females were compared to a previously published kinome dataset (11 female and 7 male subjects) which showed similar global phosphorylation signals patterns. ispartof: PLOS ONE vol:16 issue:12 ispartof: location:United States status: published

Background Based on their potential to analyze aberrant cellular signaling in relation to biological function, kinase activity profiling in tumor biopsies by peptide microarrays and mass spectrometry-based phosphoproteomics may guide selection of protein kinase inhibitors in patients with cancer. Variable tissue handling procedures in clinical practice may influence protein phosphorylation status and kinase activity and therewith may hamper biomarker discovery. Here, the effect of cold ischemia time (CIT) on the stability of kinase activity and protein phosphorylation status in fresh-frozen clinical tissue samples was studied using peptide microarrays and mass spectrometry-based phosphoproteomics. Methods Biopsies of colorectal cancer resection specimens from five patients were collected and snap frozen immediately after surgery and at 6 additional time points between 0 and 180 min of CIT. Kinase activity profiling was performed for all samples using a peptide microarray. MS-based global phosphoproteomics was performed in tumors from 3 patients at 4 time points. Statistical and cluster analyses were performed to analyze changes in kinase activity and phosphoproteome resulting from CIT. Results Unsupervised cluster analysis of kinase activity and phosphoproteome data revealed that samples from the same patients cluster together. Continuous ANOVA analysis of all 7 time points for 5 patient samples resulted in 4 peptides out of 210 (2%) with significantly (p 2) altered signal intensity in time. In 4 out of 5 patients tumor kinase activity was stable with CIT. MS-based phosphoproteomics resulted in the detection of 10,488 different phosphopeptides with on average 6044 phosphopeptides per tumor sample. 2715 phosphopeptides were detected in all samples at time point 0, of which 90 (3.3%) phosphopeptides showed significant changes in intensity with CIT (p 2. Conclusions The vast majority of the phosphoproteome as well as the activity of protein kinases in colorectal cancer resection tissue is stable up to 180 min of CIT and reflects tumor characteristics. However, specific changes in kinase activity with increasing CIT were observed. Therefore, stringent tissue collection procedures are advised to minimize changes in kinase activity during CIT.

Inherited retinal degenerative diseases (IRDs), which ultimately lead to photoreceptor cell death, are characterized by high genetic heterogeneity. Many IRD-associated genetic defects affect 3′,5′-cyclic guanosine monophosphate (cGMP) levels. cGMP-dependent protein kinases (PKGI and PKGII) have emerged as novel targets, and their inhibition has shown functional protection in IRDs. The development of such novel neuroprotective compounds warrants a better understanding of the pathways downstream of PKGs that lead to photoreceptor degeneration. Here, we used human recombinant PKGs in combination with PKG activity modulators (cGMP, 3′,5′-cyclic adenosine monophosphate (cAMP), PKG activator, and PKG inhibitors) on a multiplex peptide microarray to identify substrates for PKGI and PKGII. In addition, we applied this technology in combination with PKG modulators to monitor kinase activity in a complex cell system, i.e. the retinal cell line 661W, which is used as a model system for IRDs. The high-throughput method allowed quick identification of bona fide substrates for PKGI and PKGII. The response to PKG modulators helped us to identify, in addition to ten known substrates, about 50 novel substrates for PKGI and/or PKGII which are either specific for one enzyme or common to both. Interestingly, both PKGs are able to phosphorylate the regulatory subunit of PKA, whereas only PKGII can phosphorylate the catalytic subunit of PKA. In 661W cells, the results suggest that PKG activators cause minor activation of PKG, but a prominent increase in the activity of cAMP-dependent protein kinase (PKA). However, the literature suggests an important role for PKG in IRDs. This conflicting information could be reconciled by cross-talk between PKG and PKA in the retinal cells. This must be explored further to elucidate the role of PKGs in IRDs.

In recent years, concerns have emerged about the potential neurotoxic effects of engineered nanomaterials (NMs). Titanium dioxide and silver are among the most widely used types of metallic NMs. We have investigated the effects of these NMs on behaviour and neuropathology in male and female C57BL/6J mice following 28-day oral exposure with or without a 14-day post-exposure recovery. The mice were fed ad libitum with food pellets dosed with 10 mg/g TiO2, 2 mg/g polyvinylpyrrolidone-coated Ag or control pellets. Behaviour was evaluated by X-maze, open field, string suspension and rotarod tests. Histological alterations were analysed by immunohistochemistry and brain tissue homogenates were investigated for markers of oxidative stress, inflammation and blood-brain barrier disruption. Effects of the NMs on tyrosine and serine/threonine protein kinase activity in mouse brains were investigated by measuring kinase activity on peptide microarrays. Markers of inflammation, oxidative stress and blood-brain barrier integrity were not significantly affected in the male and female mice following exposure to Ag or TiO2. Both types of NMs also revealed no consistent significant treatment-related effects on anxiety and cognition. However, in the Ag NM exposed mice altered motor performance effects were observed by the rotarod test that differed between sexes. At 1-week post-exposure, a diminished performance in this test was observed exclusively in the female animals. Cortex tissues of female mice also showed a pronounced increase in tyrosine kinase activity following 28 days oral exposure to Ag NM. A subsequent Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) based toxicokinetic study in female mice revealed a rapid and persistent accumulation of Ag in various internal organs including liver, kidney, spleen and the brain up to 4 weeks post-exposure. In conclusion, our study demonstrated that subacute exposure to foodborne TiO2 and Ag NMs does not cause substantial neuropathological changes in mice. However, the toxicokinetic and specific toxicodynamic findings indicate that long-term exposures to Ag NM can cause neurotoxicity, possibly in a sex-dependent manner.

MotivationPhosphorylation by serine-threonine and tyrosine kinases is critical for determining protein function. Array-based approaches for measuring multiple kinases allow for the testing of differential phosphorylation between conditions for distinct sub-kinomes. While bioinformatics tools exist for processing and analyzing such kinome array data, current open-source tools lack the automated approach of upstream kinase prediction and network modeling. The presented tool, alongside other tools and methods designed for gene expression and protein-protein interaction network analyses, help the user better understand the complex regulation of gene and protein activities that forms biological systems and cellular signaling networks.ResultsWe present the Kinome Random Sampling Analyzer (KRSA), a web-application for kinome array analysis. While the underlying algorithm has been experimentally validated in previous publications, we tested the full KRSA application on dorsolateral prefrontal cortex (DLPFC) in male (n=3) and female (n=3) subjects to identify differential phosphorylation and upstream kinase activity. Kinase activity differences between males and females were compared to a previously published kinome dataset (11 female and 7 male subjects) which showed similar patterns to the global phosphorylation signal. Additionally, kinase hits were compared to gene expression databases forin silicovalidation at the transcript level and showed differential gene expression of kinases.Availability and implementationKRSA as a web-based application can be found athttp://bpg-n.utoledo.edu:3838/CDRL/KRSA/. The code and data are available athttps://github.com/kalganem/KRSA.Supplementary informationSupplementary data are available online.

About 5% of Triple negative breast cancer patients (TNBCs) who receive neoadjuvant chemotherapy (NAC) experience progressive disease (PD). Few reports are published on TNBCs with PD during NAC, whereas TNBCs that respond to NAC have been well-studied. We investigated kinase activity profiles of TNBCs to explore the biological differences underlying the lack of response to NAC. Among 740 TNBCs, 20 non-responders were identified. Seven non-responders and 10 TNBCs that did not receive NAC (control group) were evaluated. No correlation was observed between NAC response and age, menopausal status, tumor size and axillary lymph node status. Tyrosine kinase activity profiles of TNBC primary tissues from NAC non-responders and the controls were determined with a peptide microarray system. Kinase activity measurements showed that 35 peptides had significantly (p < 0.05) lower phosphorylation in non-responders. ZAP70, LCK, SYK and JAK2 were identified as differentially active upstream kinases. Pathway analysis suggested lower activity in immune-related pathways in non-responders. The number of tumor infiltrating lymphocytes (TILs) was significantly lower (p = 0.0053) in non-responders. Kinases related to the immune system are less activated in non-responders. TILs evaluation suggested that the immune system is hardly active in non-responders and is not activated by NAC treatment.

Mitogen-activated protein kinase (MAPK) inhibition with the combination of BRAF (Rapidly Accelerated Fibrosarcoma) and MEK (Mitogen-activated protein kinase kinase) inhibitors has become the standard of first-line therapy of metastatic melanoma harbouring BRAF V600 mutations. However, about half of the patients present with primary resistance while the remaining develop secondary resistance under prolonged treatment. Thus, there is a need for predictive biomarkers for sensitivity and/or resistance to further refine the patient population likely to benefit from MAPK inhibitors. In this study, we explored a top-down approach using a multiplex kinase assay, first, to discover a kinome signature predicting sensitivity, intrinsic and acquired resistance to MAPK inhibitors in melanoma, and second, to understand the mechanism of resistance using cell lines. Pre-dose tissues from patients (four responders and three non-responders to BRAFi monotherapy) were profiled for phosphotyrosine kinase (PTK) and serine-threonine kinase (STK) activities on a PamChip® peptide microarray in the presence and absence of ex vivo BRAFi. In addition, molecular studies were conducted on four sensitive parental lines, their offspring with acquired resistance to BRAFi and two lines with intrinsic resistance. PTK and STK activities in cell lysates were measured in the presence and absence of ex vivo BRAFi and/or MEKi. In tissue lysates, concentration-dependent ex vivo inhibition of STK and PTK activities with dabrafenib was stronger in responders than in non-responders. This difference was confirmed in cell lines comparing sensitive and resistant ones. Interestingly, common features of resistance were increased activity of receptor tyrosine kinases, Proto-oncogene tyrosine-protein kinase Src (Src) family kinases and protein kinase B (PKB, AKT) signalling. These latter results were confirmed by Western blots. While dabrafenib alone showed an inhibition of STK and PTK activities in both tissues and cell lines, the combination of dabrafenib and trametinib showed an antagonism on the STK activities and a synergism on PTK activities, resulting in stronger inhibitions of overall tyrosine kinase activities. Altogether; these data reveal that resistance of tumours and cell lines to MAPK inhibitors can be predicted using a multiplex kinase assay and is associated with an increase in specific tyrosine kinase activities and globally to AKT signalling in the patient’s tissue. Thus, such a predictive kinome signature would help to identify patients with innate resistance to MAPK double inhibition in order to propose other therapies.

Chemical tools and methods that report on target engagement of endogenously expressed protein kinases by small molecules in human cells are highly desirable. Here, we describe a chemical genetics strategy that allows the study of non-receptor tyrosine kinase FES, a promising therapeutic target for cancer and immune disorders. Precise gene editing was used in combination with a rationally designed, complementary fluorescent probe to visualize endogenous FES kinase in HL-60 cells. We replaced a single oxygen atom by a sulphur in a serine residue at the DFG-1 position of the ATP-binding pocket in an endogenously expressed kinase, thereby sensitizing the engineered protein towards covalent labeling and inactivation by a fluorescent probe. The temporal control offered by this strategy allows acute inactivation of FES activity both during myeloid differentiation and in terminally differentiated neutrophils. Our results show that FES activity is dispensable for differentiation of HL-60 cells towards macrophages. Instead, FES plays a key role in neutrophil phagocytosis by activation of SYK kinase, a central regulator of immune function in neutrophils. This strategy holds promise as a target validation method for kinases.

Mitogen-activated protein kinase (MAPK) inhibition with the combination of BRAF (Rapidly Accelerated Fibrosarcoma) and MEK (Mitogen-activated protein kinase kinase) inhibitors has become the standard of first-line therapy of metastatic melanoma harbouring BRAF V600 mutations. However, about half of the patients present with primary resistance while the remaining develop secondary resistance under prolonged treatment. Thus, there is a need for predictive biomarkers for sensitivity and/or resistance to further refine the patient population likely to benefit from MAPK inhibitors. In this study, we explored a top-down approach using a multiplex kinase assay, first, to discover a kinome signature predicting sensitivity, intrinsic and acquired resistance to MAPK inhibitors in melanoma, and second, to understand the mechanism of resistance using cell lines. Pre-dose tissues from patients (four responders and three non-responders to BRAFi monotherapy) were profiled for phosphotyrosine kinase (PTK) and serine-threonine kinase (STK) activities on a PamChip® peptide microarray in the presence and absence of ex vivo BRAFi. In addition, molecular studies were conducted on four sensitive parental lines, their offspring with acquired resistance to BRAFi and two lines with intrinsic resistance. PTK and STK activities in cell lysates were measured in the presence and absence of ex vivo BRAFi and/or MEKi. In tissue lysates, concentration-dependent ex vivo inhibition of STK and PTK activities with dabrafenib was stronger in responders than in non-responders. This difference was confirmed in cell lines comparing sensitive and resistant ones. Interestingly, common features of resistance were increased activity of receptor tyrosine kinases, Proto-oncogene tyrosine-protein kinase Src (Src) family kinases and protein kinase B (PKB, AKT) signalling. These latter results were confirmed by Western blots. While dabrafenib alone showed an inhibition of STK and PTK activities in both tissues and cell lines, the combination of dabrafenib and trametinib showed an antagonism on the STK activities and a synergism on PTK activities, resulting in stronger inhibitions of overall tyrosine kinase activities. Altogether; these data reveal that resistance of tumours and cell lines to MAPK inhibitors can be predicted using a multiplex kinase assay and is associated with an increase in specific tyrosine kinase activities and globally to AKT signalling in the patient’s tissue. Thus, such a predictive kinome signature would help to identify patients with innate resistance to MAPK double inhibition in order to propose other therapies.

Alzheimer’s disease (AD) is characterized by a long pre-clinical phase (20–30 years), during which significant brain pathology manifests itself. Disease mechanisms associated with pathological hallmarks remain elusive. Most processes associated with AD pathogenesis, such as inflammation, synaptic dysfunction, and hyper-phosphorylation of tau are dependent on protein kinase activity. The objective of this study was to determine the involvement of protein kinases in AD pathogenesis. Protein kinase activity was determined in postmortem hippocampal brain tissue of 60 patients at various stages of AD and 40 non-demented controls (Braak stages 0-VI) using a peptide-based microarray platform. We observed an overall decrease of protein kinase activity that correlated with disease progression. The phosphorylation of 96.7% of the serine/threonine peptides and 37.5% of the tyrosine peptides on the microarray decreased significantly with increased Braak stage (p-value

Introduction RAF-MAPK pathway inhibition with BRAF inhibitors or the combination of BRAF and MEK inhibitors has become the mainstay of therapy of metastatic melanoma harboring BRAF V600 mutations with a response rate as high as 50-75 %. A minority of patients present with primary resistance while all patients develop secondary resistance. We explored a reversed translational approach using a novel multiplex kinase assay to first identify individuals not responding to therapy using baseline biopsy samples and second to understand the mechanism of resistance using patient-derived cell lines. Method Pre-dose tissues from 4 non-responders and 3 responders to vemurafenib monotherapy, all harboring the V600E mutation, were profiled for Serine-Threonine Kinase (STK) activities on a PamChip® peptide microarray in the presence and absence of dabrafenib. For molecular studies three melanoma cell lines, all with BRAF V600E mutations, were made resistant to vemurafenib. Protein Tyrosine Kinase (PTK) and STK activities in lysates of sensitive or resistant cells were measured +/- dabrafenib, trametinib or both. Lysates were blotted against a selected panel of (phospho)proteins. Results In tissue lysates, concentration-dependent ex vivo inhibition with dabrafenib was stronger in the patients that were clinical responders than in non-responders. This difference in inhibition of STK activity by dabrafenib was confirmed in the cell lines that were made resistant against vemurafenib. IC50s for the sensitive cell lines were below 0.5 µM and varied from 10-21 µM for cell lines with acquired resistance. PTK activity was increased in resistant cell lines. STK activity was lower, but large differences exist among the cell lines. Common features of resistance were increased activity of receptor tyrosine kinases, Src family kinases and AKT signaling. These results were confirmed by Western blot analysis. Dabrafenib (10 µM) caused inhibition of STK activity while trametinib (10 µM) gave some activation. Dabrafenib (0.5 µM) gave strong inhibition of PTK activity, both in tissue and cell lines. Trametinib had hardly any inhibitory effect. Interestingly, the combination of dabrafenib and trametinib had an antagonistic effect on the STK activity and a synergistic effect on PTK activity, resulting in a stronger inhibition of kinase activity. Conclusion Resistance of tumors and cell lines to vemurafenib can affect kinase activity profiles as detected pre-dose using a multiplex kinase assay. Dabrafenib inhibition of PTK and STK activity is stronger in sensitive cell lines. The overall activation of kinases in a lysate upon MEK inhibition suggests MEK as suppressor of kinase activity. Resistance to MAPK pathway inhibitors could be linked to an increase in tyrosine kinase activity and AKT pathway activity. Biomarkers are needed to identify patients in need of combined MAPK pathway inhibition. Citation Format: Riet Hilhorst, Mohammed Krayem, Adrienne van den Berg, Fabrice Journe, Liesbeth Hovestad-Bijl, Tim van den Hooven, Rik de Wijn, Philippe Aftimos, Ghanem Ghanem, Rob Ruijtenbeek. Discovery of a kinome signature predicting sensitivity and resistance to RAF-MAPK pathway inhibitors in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3163. doi:10.1158/1538-7445.AM2017-3163

Introduction: Protein tyrosine phosphatases (PTPs) are important regulators of signal transduction in immune cells. While most analytical methods focus on the detection of these crucial enzymes at the RNA or protein level, a method was lacking to monitor multiple enzymatic activities in patient-derived materials like blood and (tumor) tissues. Such a substrate-specific assay is a new tool for biomarker discovery in immuno-oncology (I-O), because the I-O targets - checkpoint and immune receptors like PD1, CTLA4, LAG3, 4-1BB, CD40, CD20, OX40, TIGIT and GITR - are controlled by phosphatases. The aim of this study was to develop a multiplex PTP assay and investigate the phosphatase activity in metastatic renal cell carcinoma (mRCC) tissues with high and low levels of TILs (tumor-infiltrating lymphocytes). Methods: On the basis of hematoxylin and eosin staining of mRCC tissues, 9 tissues with high TILS-scores were selected, as well as 10 tissues without TILS, matched on basis of the percentage of viable tumor cells. The technology used is a second generation peptide microarray, which is fully automated with short turnaround times and a high throughput of clinical samples in a 96-well setup. Arrays comprise 46 unique nitrophosphotyrosine containing peptides derived from known phosphosites. When a clinical sample is applied to this peptide microarray, this will lead to a pattern of peptide dephosphorylations, which is monitored in real time (kinetic readout). Results: The PTP assay was first developed for PBMC profiling. 0.10 ug protein/array was used, corresponding to 10000 cells. The signals were concentration dependent and could be inhibited by PTP inhibitors. For tissue profiling 2.0 µg protein input (0.02 mm3 tissue) was used. A 2-group comparison analysis of the phosphatase activity in tissues with high scores of TILs (massive infiltration, N=9) vs. without (N=10), revealed that 29 of 46 peptide substrates showed significantly higher dephosphorylation (PTP) activities in the tissues with high levels of lymphocyte infiltrates. A PCA revealed a clear separation between the groups of high and low TILS scores. Discriminating phosphosites (p Conclusions: Proof of concept was shown in mRCC samples that high PTP activity correlates with high levels of TILS. As a TILS-score itself is already regarded to be a candidate biomarker for I-O therapy response, the observed correlation is a basis for the development of more mechanistic biomarkers predicting therapy response. This method can be a starting point for the development of an enzymatic and thus sensitive and quantitative TILS test. This study has received funding from the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no 259939. Citation Format: Rob Ruijtenbeek, Liesbeth Houkes-van Kerkhoff, Jeannette Oosterwijk, Liesbeth Hovestad-Bijl1, Riet Hilhorst, Alejandro H. Gomez, Juan F. Rodriguez, Jesus Garcia-Donas, Bart Kiemeney, Egbert Oosterwijk. Correlation of phosphatase activity with lymphocyte infiltrates in metastatic renal cell carcinoma tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5608. doi:10.1158/1538-7445.AM2017-5608

Background: Triple-negative breast cancer patients (TNBCs) who receive neoadjuvant chemotherapy (NAC) generally show a response to NAC, but about 5% experience progressive disease (PD). Although TNBCs that respond to NAC have been well-studied, reports about TNBCs with PD during NAC are absent. We aimed to compare kinase activity profiles of TNBCs of both groups to identify a biological predictor of NAC non-responders. Methods: Tyrosine kinase activity profiles of lysates of fresh frozen cancer tissues from NAC non-responders and responders, the latter represented by TNBCs who did not receive NAC, were determined with a peptide microarray system (Cancer Res. 2009; 69(14):5987-95). Tumor infiltrating lymphocytes (TILs) were counted in these tissues. Results: Among 740 TNBCs, 20 NAC non-responders were identified. For the 7 samples from PD confirmed patients and 10 TNBCs not receiving NAC, no correlation was observed between NAC response and age, menopausal status, tumor size or axillary lymph node status. Kinase activity measurements showed large differences in overall tyrosine kinase activity between responders and non-responders. 35 peptides had significantly (p < 0.05) higher phosphorylation in the responder group. Kinases that phosphorylate these peptides (upstream kinases) were identified from literature. Among others, the immune related kinases Zap70, Syk and JAK2 were identified as upstream kinases. Pathway analysis yielded evidence for increased activity in the responder group in immune-related pathways and in pathways involved in development and signal transduction. Responders had a significantly higher (p = 0.0053) TILs count. Conclusion: Kinases related to the immune system are less activated in NAC non-responders. The low TILs count in the non-responders confirmed that activation of the immune system in non-responders is lower than in NAC responders. These data merit a further investigation of the use of kinase activity profiling either alone or in combination with TIL scoring for the prediction of the response to NAC in TNBC with a larger patient cohort. Citation Format: Takeshi Sawada, Riet Hilhorst, Savithri Rangarajan, Masayuki Yoshida, Kenji Tamura, Rinie van Beuningen, Rob Ruijtenbeek, Hitoshi Tsuda, Fumiaki Koizumi. Immune system pathway activation for prediction of the response to neoadjuvant chemotherapy in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1792. doi:10.1158/1538-7445.AM2017-1792

The tyrosine-containing peptide Gly-Tyr-Gly (GYG) was oxidatively cross-linked by horseradish peroxidase in the presence of hydrogen peroxide. As products, covalently coupled di- to pentamers of the peptide were identified by LC-MS. Oxidative cross-linking of ferulic acid with horseradish peroxidase and hydrogen peroxide resulted in the formation of dehydrodimers. Kinetic studies of conversion rates of either the peptide or ferulic acid revealed conditions that allow formation of heteroadducts of GYG and ferulic acid. To a GYG-containing incubation mixture was added ferulic acid in small aliquots, therewith keeping the molar ratio of the substrates favorable for heterocross-linking. This resulted in a predominant product consisting of two ferulic acid molecules dehydrogenatively linked to a single peptide and, furthermore, two ferulic acids linked to peptide oligomers, ranging from dimers to pentamers. Also, mono- and dimers of the peptide were linked to one molecule of ferulic acid. A mechanism explaining the formation of all these products is proposed. Chemicals/CAS: Coumaric Acids; Cross-Linking Reagents; ferulic acid, 1135-24-6; Horseradish Peroxidase, EC 1.11.1.-; Hydrogen Peroxide, 7722-84-1; Tyrosine, 55520-40-6

Curculigo pilosa is traditionally used in the manufacture of sorghum beer in West Africa. β-Amylase was purified 100-fold with 38% yield from a crude extract, giving final specific activities of 4850 U/mg and 5650 U/mg using soluble starch and p-nitrophenyl maltopentaoside, respectively, as substrates. The molecular mass of the monomeric enzyme was 64 kDa and its pI 4.2. Both activity and thermostability are higher than reported for other plant β-amylases. The catalytic efficiency was lower for amylose than for starches and amylopectin. In contrast to other plant amylases, the β-amylase from C. pilosa is able to degrade raw starches from wheat, corn, potato and rice. In this respect, it resembles β-amylases from microbial origin. This property, and its high activity and stability, explain its traditional use in the manufacture of infant food and sorghum beer in Burkina Faso and could make it applicable for other biotechnological purposes.

Alzheimer’s disease (AD) is characterized by the deposition of amyloid-β (Aβ), which is associated with a neuroinflammatory response involving microglia and astrocytes. This neuroinflammatory response has detrimental effects on disease progression but also has a beneficial function on removal of excess Aβ. Microglia and astrocytes are involved in the clearance of Aβ from the brain, but neuroinflammation also promotes neurodegeneration. In order to identify signal transduction pathways critically involved in AD we analysed human brain tissue using protein kinase activity profiling. We identified increased activity of the Interleukin 1 Receptor Associated Kinase 4 (IRAK-4) in AD compared to control brain tissue. IRAK-4 is a component of the signal transduction pathway that functions downstream of the Toll-like receptors and the interleukin-1 receptor. Immunohistochemical analysis of human brain tissue revealed the presence of IRAK-4 in astrocytes and microglia. Quantification of IRAK-4 and the phosphorylated form of IRAK-1, a specific substrate for IRAK-4, revealed increased expression and activity of IRAK-4 in AD. Interestingly, IRAK-1/4 inhibitor I reduces the lipopolysaccharide-induced secretion of monocyte chemotactic protein-1 (MCP-1) by primary human microglia and the interleukin-1β-induced secretion of MCP-1 and interleukin 6 by primary human astrocytes. In contrast, the uptake of Aβ by astrocytes and microglia is not affected by IRAK-1/4 inhibition. Our data show that IRAK-4 protein kinase activity is increased in AD and selective inhibition of IRAK-1/4 inhibits a pro-inflammatory response without affecting the uptake of Aβ by glial cells, indicating that the IRAK signalling pathway is a potential target for modulating neuroinflammation in AD.

Background: Humoral immunity related to neuronand disease-specific cytoskeletal proteins in patients with Alzheimer’s disease (AD) is largely unknown. Methods: Antibodies against three targets [tau, light (NFL) and heavy (NFH) neurofilaments] were measured using ELISA in 80 paired serum and CSF samples from 25 patients with AD fulfilling NINCDSADRDA criteria, 13 patients with other dementias (OD), 25 cognitively normal elderly controls (NC), 17 patients with neuroinflammatory diseases (IC). We estimated intrathecal synthesis of all antibodies according to formula (CSF anti-neurocytoskeletal IgG /serum anti-neurocytoskeletal IgG)/ (CSF total IgG /serum total IgG).Results:TheAD patients had significantly higher intrathecal anti-tau antibodies than in the OD group (p 1⁄4 0.005) or the IC group (P 1⁄4 0.0003) and higher intrathecal anti-NFH antibodies than in the OD group (P 1⁄4 0.046), the NC group (p 1⁄4 0.04) or the IC group (P 1⁄4 0.0007), but not intrathecally synthesized anti-NFL antibodies. All three anti-neurocytoskeletal antibodies in serum or CSF did not differ among four groups with the exceptions for the IC group having them elevated in CSF. Conclusions: These new findings suggest some hints of specific alterations in humoral anti-neurocytoskeletal immunity and selectivity in AD. It may have diagnostic and immunotherapeutic implications.

Janus kinase 2 (JAK2) initiates signaling from several cytokine receptors and is required for biological responses such as erythropoiesis. JAK2 activity is controlled by regulatory proteins such as Suppressor of Cytokine Signaling (SOCS) proteins and protein tyrosine phosphatases. JAK2 activity is also intrinsically controlled by regulatory domains, where the pseudokinase (JAK homology 2, JH2) domain has been shown to play an essential role. The physiological role of the JH2 domain in the regulation of JAK2 activity was highlighted by the discovery of the acquired missense point mutation V617F in myeloproliferative neoplasms (MPN). Hence, determining the precise role of this domain is critical for understanding disease pathogenesis and design of new treatment modalities. Here, we have evaluated the effect of inter-domain interactions in kinase activity and substrate specificity. By using for the first time purified recombinant JAK2 proteins and a novel peptide micro-array platform, we have determined initial phosphorylation rates and peptide substrate preference for the recombinant kinase domain (JH1) of JAK2, and two constructs comprising both the kinase and pseudokinase domains (JH1-JH2) of JAK2. The data demonstrate that (i) JH2 drastically decreases the activity of the JAK2 JH1 domain, (ii) JH2 increased the Km for ATP (iii) JH2 modulates the peptide preference of JAK2 (iv) the V617F mutation partially releases this inhibitory mechanism but does not significantly affect substrate preference or Km for ATP. These results provide the biochemical basis for understanding the interaction between the kinase and the pseudokinase domain of JAK2 and identify a novel regulatory role for the JAK2 pseudokinase domain. Additionally, this method can be used to identify new regulatory mechanisms for protein kinases that provide a better platform for designing specific strategies for therapeutic approaches. Public Library of Science

Triple-negative breast cancers (TNBC), characterized by absence of estrogen receptor (ER), progesterone receptor (PR) and lack of overexpression of human epidermal growth factor receptor 2 (HER2), are typically associated with poor prognosis, due to aggressive tumor phenotype(s), only partial response to chemotherapy and present lack of clinically established targeted therapies. Advances in the design of individualized strategies for treatment of TNBC patients require further elucidation, by combined ‘omics’ approaches, of the molecular mechanisms underlying TNBC phenotypic heterogeneity, and the still poorly understood association of TNBC with BRCA1 mutations. An overview is here presented on TNBC profiling in terms of expression signatures, within the functional genomic breast tumor classification, and ongoing efforts toward identification of new therapy targets and bioimaging markers. Due to the complexity of aberrant molecular patterns involved in expression, pathological progression and biological/clinical heterogeneity, the search for novel TNBC biomarkers and therapy targets requires collection of multi-dimensional data sets, use of robust multivariate data analysis techniques and development of innovative systems biology approaches.

The rate of mass transfer in the liquid—liquid extraction of the enzyme α-amylase between an aqueous phase and a reversed micellar phase has been investigated. Mass transfer rate coefficients have been measured in a mixer/settler and in a stirred cell. The pH of the aqueous phase determines the distribution coefficient of the enzyme and thus the direction of transfer. Forward transfer of the enzyme from the aqueous to the reversed micellar phase (cationic surfactant) occurs at pH 10.0. The mass transfer rate of this process was found to be controlled by the diffusion of the enzyme in the aqueous phase boundary layer. Back transfer from the reversed micellar phase to the aqueous phase occurs in the pH range 4–6. In contrast to the forward transfer, this process was found to be controlled by the interfacial process of enzyme release from the reversed micelles instead of the boundary layer diffusion. The same effects have been observed for the transfer of the enzyme ribonuclease A to and from a reversed micellar phase with an anionic surfactant. A mechanism to explain the different mass transfer behaviour in forward and back transfer is suggested.

The major cationic peroxidase in sorghum grain (SPC4) , which is ubiquitously present in all sorghum varieties was purified to apparent homogeneity, and found to be a highly basic protein (pI approximately 11). MS analysis showed that SPC4 consists of two glycoforms with molecular masses of 34,227 and 35,629 Da and it contains a type-b heme. Chemical deglycosylation allowed to estimate sugar contents of 3.0% and 6.7% (w/w) in glycoform I and II, respectively, and a mass of the apoprotein of 33,246 Da. High performance anion exchange chromatography allowed to determine the carbohydrate constituents of the polysaccharide chains. The N-terminal sequence of SPC4 is not blocked by pyroglutamate. MS analysis showed that six peptides, including the N-terminal sequence of SPC4 matched with the predicted tryptic peptides of gene indice TC102191 of sorghum chromosome 1, indicating that TC102191 codes for the N-terminal part of the sequence of SPC4, including a signal peptide of 31 amino acids. The N-terminal fragment of SPC4 (213 amino acids) has a high sequence identity with barley BP1 (85%), rice Prx23 (90%), wheat WSP1 (82%) and maize peroxidase (58%), indicative for a common ancestor. SPC4 is activated by calcium ions. Ca2+ binding increased the protein conformational stability by raising the melting temperature (Tm) from 67 to 82 degrees C. SPC4 catalyzed the oxidation of a wide range of aromatic substrates, being catalytically more efficient with hydroxycinnamates than with tyrosine derivatives. In spite of the conserved active sites, SPC4 differs from BP1 in being active with aromatic compounds above pH 5.

Ethnobotanical and biochemical surveys revealed that some local plants from West Africa are novel sources of polysaccharide degrading enzymes such as amylases and glucanases. The study shows that these enzymes could be used for various biotechnological applications. In a crude extract ofCurculigo pilosa, ß-amylase was the main starch hydrolyzing enzyme. Contrary to other plant amylases, the ß-amylase from C. pilosa is able to degrade raw starches from wheat, corn, potato and rice. In the bulbs of Gladiolus klattianus, activities of a-amylase and ß-amylase were found. Analysis of the enzyme action pattern showed that it released only maltose units from starch. Activities of aamylase, ß-amylase, exo-(1®3, 1®4)-ß-D-glucanase and endo-(1®3)-ß-D-glucanase were detected in the leaves of Boscia senegalensis. The combined action of a saccharogenic enzyme (ß-amylase) and a dextrinizing enzyme (a-amylase) in B. senegalensis was useful to decrease the viscosity of cerealporridges and to increase their reducing sugar contents. The effective technological utilization of these higher plants as sources of carbohydrate degrading enzymes is discussed.

Ferulic acid (FA) is an abundantly present phenolic constituent of plant cell walls. Kinetically controlled incubation of FA and the tripeptide Gly-Tyr-Gly (GYG) with horseradish peroxidase and H2O2 yielded a range of new cross-linked products. Two predominant series of hetero-oligomers of FA linked by dehydrogenation to the peptidyl tyrosine were characterized by electrospray ionization (tandem) mass spectrometry. One series comprises GYG coupled with 4-7 FA moieties linked by dehydrogenation, of which one is decarboxylated. In the second series 4-9 FA moieties linked by dehydrogenation, of which two are decarboxylated, are coupled to the tripeptide. A third series comprises three heterooligomers in which the peptidyl tyrosine is linked to 1-3 FA moieties of which none is decarboxylated. Two mechanisms for the formation of the FA-Tyr oligomers that result from the dualistic, concentration-dependent chemistry of FA and their possible role in the regulation of plant cell wall tissue growth are presented. Chemicals/CAS: Coumaric Acids; ferulic acid, 1135-24-6; Glycine, 56-40-6; Horseradish Peroxidase, EC 1.11.1.-; Hydrogen Peroxide, 7722-84-1; Oxygen, 7782-44-7; Peptides; Tyrosine, 55520-40-6

Background: There is an urgent need to develop companion diagnostics for response prediction to increase the success rate of anticancer drug development. Constitutively activated protein kinases are major targets for cancer treatment. However, blockade of target molecules may not necessarily lead to treatment response. Thus we attempted to investigate the consequences of target kinase inhibition by comprehensive tyrosine kinases activity profiling, which can be applied to establish a response prediction signature. Here we employed clinically validated EGFR-targeted inhibition as in vitro model utilizing PamChip® peptide micro-arrays. Methods: Eight lung cancer cell lines harboring either wild-type EGFR (A549, H1781 and H441) or mutant EGFR (HCC4006, PC9, HCC827, H1650 and H1975) were used. Chemosensitivity to EGFR inhibitors (gefitinib, erlotinib and afatinib) was evaluated by MTT assay and IC50 values were obtained. For assessment of protein tyrosine kinase activity, cell lysates were prepared, aliquoted and stored at -80°C until use. The tyrosine kinase activity of the lysates was measured with or without EGFR TKIs on PamChip® peptide micro-arrays, containing 144 peptides derived from known human phosphorylation sites. Data analysis was carried out on both basal and inhibition profiles. Inhibition profiles were calculated for each EGFR TKI by taking the log-ratio of peptide phosphorylation measured with or without EGFR TKIs using BioNavigator6 software. Peptides that did not show increase of signal in time (indicative of kinase activity) were excluded from the analysis. Results: Basal tyrosine kinase activity profiles showed a clear segregation between EGFR driven (HCC4006, PC9, and HCC827) and EGFR independent (H1650, H441 and A549) cells. Additionally, overall tyrosine kinase activities in EGFR mutant cells were higher than those in EGFR wild-type cells. Based on IC50 values for three EGFR TKIs, the cell lines were divided into responders (PC9, HCC4006 and HCC827 with IC50 values ≦0.1 μmol/L), non-responders (H1650, H441 and A549 with IC50 values >1 μmol/L) and intermediate responders. Basal phosphorylation signals on a number of peptides differed between responders and non-responders. The responders showed phosphorylation on sites responsible for activating RAF, ERK1/2 and MAPK7 and on sites for inactivating the phosphatases PP2AB and PTN11. The phosphorylation was absent in the non-responders, possibly as a consequence of constitutive activation of the RAS/RAF pathway. This peptide set was suggested to serve as a predictive signature of response to EGFR TKIs. Conclusions: Kinase activity profiles reflect the biochemical consequences of EGFR mutant non-small-cell lung cancer. Tyrosine kinase activity profiling may serve as companion diagnostic in drug development and this should be further explored. Citation Format: Naoki Izawa, Masakuni Serizawa, Faris Naji, Rik de Wijn, Riet Hilhorst, Rob Ruijtenbeek, Hirofumi Yasui, Takako Nakajima, Narikazu Boku, Yasuhiro Koh. Predictive signature of response to EGFR tyrosine kinase inhibitors in non-small-cell lung cancer cells by tyrosine kinase activity profiling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4248. doi:10.1158/1538-7445.AM2014-4248

Background High-grade osteosarcoma is a primary malignant bone tumor mostly occurring in adolescents and young adults, with a second peak at middle age. Overall survival is approximately 60%, and has not significantly increased since the introduction of neoadjuvant chemotherapy in the 1970s. The genomic profile of high-grade osteosarcoma is complex and heterogeneous. Integration of different types of genome-wide data may be advantageous in extracting relevant information from the large number of aberrations detected in this tumor. Methods We analyzed genome-wide gene expression data of osteosarcoma cell lines and integrated these data with a kinome screen. Data were analyzed in statistical language R, using LIMMA for detection of differential expression/phosphorylation. We subsequently used Ingenuity Pathways Analysis to determine deregulated pathways in both data types. Results Gene set enrichment indicated that pathways important in genomic stability are highly deregulated in these tumors, with many genes showing upregulation, which could be used as a prognostic marker, and with kinases phosphorylating peptides in these pathways. Akt and AMPK signaling were identified as active and inactive, respectively. As these pathways have an opposite role on mTORC1 signaling, we set out to inhibit Akt kinases with the allosteric Akt inhibitor MK-2206. This resulted in inhibition of proliferation of osteosarcoma cell lines U-2 OS and HOS, but not of 143B, which harbors a KRAS oncogenic transformation. Conclusions We identified both overexpression and hyperphosphorylation in pathways playing a role in genomic stability. Kinome profiling identified active Akt signaling, which could inhibit proliferation in 2/3 osteosarcoma cell lines. Inhibition of PI3K/Akt/mTORC1 signaling may be effective in osteosarcoma, but further studies are required to determine whether this pathway is active in a substantial subgroup of this heterogeneous tumor.

Single-chain Fv antibody fragments binding different flavin forms [10-(5'-carboxybutyl-)flavin (Fl[ox]) and 10-(5'-carboxybutyl)-1,5-dihydroflavin (Fl[red])] have been generated from an antibody phage-display library to study how a protein environment regulates the redox potential, starting from a protein other than a natural flavoprotein. These 'flavobodies' are characterized by time-resolved and steady-state fluorescence spectroscopy, by competitive ELISA methods (mapping of the antigen-binding site), and by molecular modelling. The three-dimensional models of the antigen-binding sites are consistent with the experimental results. Binding of anti-Fl(red) 5 to flavin increases the redox potential, mainly due to an Arg residue interacting with the flavin N1. Thus anti-Fl(red) 5 shows an 'oxidase-like' redox-potential behaviour, confirming the idea that positively charged residues in the vicinity of N1 increase the redox potential. The results obtained with anti-Fl(ox), which do not resemble a natural flavoprotein, show that when the pyrimidine-like nucleus of the flavin is not involved in binding, the redox potential is not significantly affected. These results are in contrast to those obtained with chicken riboflavin-binding protein.

Background: Gastric cancer is among the leading causes of cancer-related deaths worldwide, notably in Asia. Though targeted therapies have been under clinical evaluation, the biological complexity of this cancer type may confer resistance to single-targeted therapies. Especially, scirrhous phenotype is known to be affected by multiple growth factors. To overcome this biological complexity, multi-targeted kinase inhibitors (MTKIs) have been clinically tested and were reported to be effective in a subset of patients. We wanted to elucidate the kinase activity signature of gastric cancer and to explore predictive biomarkers of MTKIs using gastric cancer cell lines by tyrosine kinase activity profiling. Material and methods: The growth-inhibitory effect of the MTKIs sunitinib, sorafenib, pazopanib and the Akt inhibitor MK-2206 was evaluated by the MTT assay. 11 scirrhous and 14 non-scirrhous gastric cancer cell lines were grown until semi-confluent, lysed, aliquoted and stored at -80°C until use. The basal tyrosine kinase activity of the lysates was measured on PamChip® peptide micro-arrays, containing 144 peptides derived from known human phosphorylation sites. Quantification of peptide phosphorylation and data analysis were performed using BioNavigator and SIMCA P+ software. Results: HSC-39, HSC-40A, KATO-III and HSC-43 scirrhous cells and SNU-16 non-scirrhous cells were sensitive to all three MTKIs with IC50 values < 1 μmol/L. Orthogonal partial least square-discriminant analysis (OPLS-DA) of basal tyrosine kinase activity profiles showed a clear segregation between sensitive cell lines and insensitive cell lines. Levels of putative target proteins such as PDGFR, VEGFR, Raf and Kit did not correlate with the sensitivity. All sensitive cell lines turned out to harbor an FGFR2 amplification suggesting that FGFR2 amplification may be a predictive biomarker of response to MTKI treatment. Kinases such as EGFR, EPHA, FGFR, IGF1R, INSR and Src contributed to this segregation suggesting that these are relevant to the sensitivity to MTKIs. MET overexpressing cell lines were resistant to treatment with the four inhibitors. Scirrhous cells were segregated from non-scirrhous cells by OPLS-DA among cell lines with signet-ring cell carcinoma cytology with R2, 0.994; Q2, 0.615. EGFR, ERBB2, 3, 4, EPHAs, FAK, IGF1R, INSR, MAP2Ks, RET and SRC seemed to be responsible for the segregation suggesting that they play an important role in scirrhous gastric cancer biology. Conclusions: Kinase activity profiles reflect the biological complexity of gastric cancer. FGFR2 status correlates with sensitivity to sunitinib, pazopanib and sorafenib, whereas MET status does not. These data imply that tyrosine kinase activity profiling with PamChip® peptide arrays can be utilized to identify molecular targets and predictive biomarkers in gastric cancer and this should be further explored. Citation Format: Yasuhiro Koh, Masakuni Serizawa, Rik de Wijn, Riet Hilhorst, Takako Nakajima, Hirofumi Yasui, Kazuyoshi Yanagihara, Martijn Dankers, Rob Ruijtenbeek, Narikazu Boku. In vitro tyrosine kinase activity profiling to identify molecular targets and predictive biomarkers in gastric cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4655. doi:10.1158/1538-7445.AM2013-4655

The galectin protein family consists of fifteen galactoside-binding proteins that are present in all classes of life where they function as cell-cell and cell-matrix interaction mediators. Galectins are however also highly overexpressed in various processes related to cancer, including apoptosis, angiogenesis and metastasis. Galectins are therefore excellent targets for tumor diagnostics and medical prognostic purposes. Current proteomic tools, e.g. Polymerase Chain Reaction (PCR) and Enzyme-linked Immunosorbent Assay (ELISA), are based on protein abundance rather than protein activity. In contrast, we have developed a galectin detection assay that visualizes galactoside-binding, i.e. functional, galectins using PamChip® microarray technology. Firstly, antibodies directed against different galectins are efficiently immobilized on the microarray surface by the Fc-binding protein A/G. Then galectin-1 was captured from a spiked cell lysate that is added to the microarray and the unbound proteins are washed away. The galectin presence is subsequently quantified with a general fluorescent ligand that is only bound by active galectins. Several ligands based on lactose and lactosamine were designed and tested. With the best ligands it was possible to detect galectin quantities in the low nanogram range using only 1 μg or less of precious anti-galectin antibodies per measurement. These results are truly promising for the use of the newly developed galectin detection technology in a clinical setting. Citation Format: Hilde van Hattum, Rob Ruijtenbeek, Riet Hilhorst, Roland Pieters. Development of a new and sensitive detection technology for galectin cancer proteins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4136. doi:10.1158/1538-7445.AM2013-4136

Background. Due to the biological heterogeneity, response to systemic therapy is variable in colon cancer. Chemotherapy improves median survival from 6-12 months to 18-24 months. Epidermal growth factor receptor (EGFR) antibodies have shown to improve survival, although modest, even further. KRAS mutations were identified as negative predictive markers for EGFR directed therapy. The response rate to EGFR targeted therapy remains low. Therefore there is an urgent need to find additional therapeutic options and accompanying biomarkers. The analysis of kinase activity in colon tumors may yield tumor-specific information on aberrant cell signaling pathways and may be usefull in a better patient stratification. Aim of this study is to generate overall kinase activity profiles from colon cancer tissues to explore if subtypes of this cancer show distinct kinase activity profiles. This can help to find new therapeutic options or predictive markers for response to therapy. Methods. Normal and tumorous colon tissue from patients who had given informed consent was fresh frozen after surgical resection. All tumor specimens were analyzed for BRAF and KRAS mutation status. Tissue cryosections were lysed in buffer with phosphatase and protease inhibitors. Tyrosine kinase (PTK) and serine/threonine kinase (STK) activity profiles of normal and tumor tissue were generated on PamChip® peptide microarrays comprising peptide sequences derived from known human phosphorylation sites. The ex vivo effect of kinase inhibitors on these kinase activity profiles was also determined. Peptide phosphorylation was monitored using fluorescently labeled antibodies. Data were analysed with Bionavigator software. Results. Tumor tissue has a higher PTK activity than normal tissue, whereas STK activity was equal to or lower than in normal tissue. STK activity profiles of 23 colon tumors could not be grouped according to mutation status, percentage of tumor tissue, differentiation grade or TNM classification. The STK activity profiles showed that within the group of wild type KRAS tumors, clearly different clusters were found. In the group with KRAS mutations (G12V, G12D, G12C, G12R) the STK activity profiles differed between the tumors. A subset of tumors, (two KRAS mutants, a BRAF mutant and a WT tumor) were tested ex vivo with a selection of 11 kinase inhibitors and showed different sensitivity to these inhibitors. Conclusions. Kinase activity and the response to inhibitors can be measured ex vivo in colon tumor tissue to capture the diversity in kinase activity and response to inhibitors between patients. The differences in kinase activity profiles of colon cancer samples could not be explained by subtypes according to mutation (KRAS, BRAF) status. Interesting subclusters could be identified using the kinase activity profiling providing a higher granularity than KRAS mutation scoring allows. Citation Format: Rosanne van den Oord, Hans Pruijt, Adriaan van den Brule, Peet Nooijen, Rik de Wijn, Monique Mommersteeg, Riet Hilhorst, Rob Ruijtenbeek. Interpatient differences in colon tumors reflected in kinase activity and inhibition profiles related to KRAS and BRAF mutation status. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5586. doi:10.1158/1538-7445.AM2013-5586

A procedure is presented to quantitatively estimate the growth of a particular organism in a food product during chilled storage using predictive microbiology. This results in a quantification of the contribution of every individual process step to the total number of organisms, which may be a useful tool to support decisions on existing process lines as well as in process and product design. It is demonstrated that predictive microbiology will only estimate to within orders of magnitude of bacterial growth. This helps to pinpoint the most important aspects of a line. The calculations can be helpful to set critical limits and to detect hazards by performing 'what if' analyses. The procedure is explained for the growth of Bacillus cereus in milk. It is indicated, that with the current information, the effect of time/temperature can be estimated. However, to make an accurate exposure analysis, more information will be needed.

In order to create a protein environment that binds preferentially to the two-electron reduced form of flavin, monoclonal antibodies have been raised against a reduced flavin derivative. Due to the low fluorescence quantum yield and visible light absorption and to the instability of reduced flavin in an aerobic environment, it is not possible to determine the affinities of these antibodies for two-electron-reduced flavin using standard techniques. Because of its sensitivity, time-resolved fluorescence can be used to overcome this problem. This technique has been applied to study the binding of two antibodies, an IgG1 and an IgM, to reduced riboflavin (1,5-dihydroriboflavin) and oxidized riboflavin (riboflavin). The affinity of the IgG1 is more than 80 times larger for 1,5-dihydroriboflavin than for riboflavin. From analysis of the dynamical parameters of the system it is apparent that the internal motion of 1,5-dihydroriboflavin bound to IgG1 is much more restricted than that of riboflavin. In contrast, the affinity of the IgM is only slightly higher for 1,5-dihydroriboflavin than for riboflavin and the flexibility of binding of both flavin redox states in the antigen binding site is almost similar.

Background. Whereas ALK translocations play a role in tumor growth in selected NSCLC cases, mutated forms of ALK have been shown to drive development of neuroblastoma, the most common malignant extracranial tumor of childhood. The mutation F1147L in the oncogene ALK increases the kinase activity, causing deregulated proliferation of sympatho-adrenal progenitor cells. We explored the use of kinase activity measurements to identify ALK activity in cell lysates and patient derived tissue lysates with the aim of identifying biomarkers to trace the activity of ALK in tumors and the effect of ALK inhibitors on these marker signals. Methods. Protein kinase activities were measured on PamChip® peptide microarrays comprising 144 Tyr containing peptide sequences from known human phosphorylation sites. The activity profiling experiments involved recombinant ALK kinase, lysates of cell lines overexpressing ALK and neuroblastoma tumors with elevated ALK F1174L presence. Per microarray analysis 5 ug of total protein was used in the presence of 100 uM ATP. Experiments were performed in the presence and absence of ALK kinase inhibitors crizotinib and NVP-TAE684. Real time kinetics of 144 peptide phosphorylations were measured with a fluorescently labeled antibody. Results. Phosphorylation profiles for neuroblastoma tumors, ALK overexpressing cell lines and recALK were generated. For recALK 48 novel peptide substrates were identified that showed signals depending on both ATP and kinase concentration and inhibition by the ALK inhibitors. In ALK overexpressing cell lines, but not in control cell lysates, signals on many ALK substrate peptides were significantly elevated, and returned to baseline level in the presence of ALK inhibitors. Whereas neuroblastomas with low ALK expression gave similar kinase activity profiles, the tumors harboring the ALK F1174L mutation were different, suggesting a more complex regulation of phosphorylation activity in these tumors. All tumors showed on-chip responses with ALK inhibitor drugs, but to different extent, which could be a basis for response prediction biomarker discovery. Conclusion. ALK activity of recombinant kinases, cell lysates and tumor lysates can be detected on PamChip® peptide microarrays. Based on results with model systems, this method shows promise to identify tumors that express elevated ALK activity and to study response of this class to ALK inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1683. doi:1538-7445.AM2012-1683

Background: Reliable diagnostic tests are needed to identify early stage non-small cell lung carcinoma (NSCLC) patients with poor prognosis. Concomitantly there is a clear need for tests that enable the selection of patients who will benefit from targeted therapy with kinase inhibitors. We evaluated kinase activity profiles in two groups of early stage NSCLC patients, either for prognosis of long- or short-term survival, or for predicting erlotinib drug response. Method: Retrospective studies were performed on fresh frozen resection material of two groups of early stage NSCLC patients. The first group consisted of 48 short- and long-term survivors who underwent a complete surgical resection (5+ years follow-up). The second group consisted of 14 NSCLC patients who received 3 weeks of neo-adjuvant treatment with erlotinib prior to complete surgical resection. Response evaluation to neo-adjuvant treatment was based on histopathological examination of the surgical specimens. For both studies, kinase activity profiles of lysed cryosections of tumour tissues were generated in the presence and absence of protein tyrosine kinase inhibitors on PamChip® peptide micro-arrays, comprising 144 tyrosine containing peptides, derived from known phosphorylation sites of human proteins. Partial least square discriminant analysis was used to construct prediction models. ClustalW alignment algorithms were used to investigate the most informative phosphorylation sites. Results: Kinase activity profiles obtained in the absence of inhibitor did not distinguish between subgroups (long- versus short-term survival, responder or non-responder to TKI), whereas ratios of inhibited versus non-inhibited signals resulted in distinct classifiers predicting survival for the first group, and response for the second group. Multivariate unsupervised analysis with leave-one-out cross-validation resulted in an error rate for survival prediction of 29%. In the drug response prediction 13 out of 14 patients were correctly classified. Conclusion: This is the first study to show that kinase activity profiles of tumour tissue exposed to a kinase inhibitor can be used to identify NSCLC patients likely to respond to erlotinib treatment. Furthermore, based on kinase activity profiles of early stage NSCLC tumours, a prognostic classifier, for a set of 48 patients, was obtained. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4113. doi:10.1158/1538-7445.AM2011-4113

Scirrhous-type gastric cancer, which accounts for about 10% of all gastric cancers, interacts with stromal cells with involvement of multiple growth factors. Although a fluoropyrimidine and platinum-containing regimen has been established as the standard chemotherapy, its efficacy is not satisfactory. Multi-targeted kinase inhibitors (MTKIs) such as sunitinib, sorafenib and pazopanib are active in renal cell carcinoma and are under clinical evaluation for other types of solid tumors. Here we examined the efficacy of the MTKIs in scirrhous gastric cancer cell lines and conducted an exploratory predictive biomarker study by tyrosine kinase activity profiling with PamChip® peptide micro-arrays. For 11 scirrhous and 8 non-scirrhous gastric cancer cell lines, the growth-inhibitory effect of the MTKIs sunitinib, sorafenib and pazopanib was evaluated by the MTT assay. For assessment of protein tyrosine kinase activity, cells were grown until semi-confluent, lysed, aliquoted and stored at -80°C until use. The tyrosine kinase activity of the lysates was measured in the absence and presence of the MTKIs on PamChip® peptide micro-arrays, containing 144 peptides derived from known human phosphorylation sites. Data analysis was carried out on inhibition profiles calculated for each MTKI by taking the log-ratio of peptide phosphorylation measured in the absence and presence of the MTKIs. Peptides with a low basal signal were excluded from the analysis. In the MTT assay, cell lines HSC-39, HSC-40A, KATO-III, HSC-43 (scirrhous) and SNU-16 (non-scirrhous) were sensitive to all three MTKIs with IC50 values < 1 μmol/L. The other 14 cell lines were classified as insensitive, except that three cell lines showed intermediate sensitivity to sunitinib with IC50 values < 3 μmol/L. Per peptide analysis showed a clear correlation between sensitivity to the MTKIs and in vitro inhibition of tyrosine kinase activity, with stronger response associated with more inhibition for a larger number of peptides (out of 100 peptides 48, 46, and 30 with p < 0.05 for sunitinib, sorafenib and pazopanib, respectively). Principal component analysis of the inhibition profiles revealed a clustering of sensitive vs. insensitive cell lines. Partial least squares-discriminant analysis was used to correlate the in vitro inhibition on the PamChip® micro-arrays to the sensitivity to the MTKIs. Good predictive performance with a misclassification rate < 20% was obtained with leave-one-out-cross-validation for all 3 inhibitors. MTKIs seem to be efficacious in a subset of scirrhous-type gastric cancer. Sunitinib showed the most potent growth-inhibitory effect, suggesting that it should be considered for further evaluation in scirrhous-type gastric cancer. Sensitivity to the MTKIs in the MTT assay could be correlated to the in vitro inhibition of kinase activity on the PamChip® peptide micro-array. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4468. doi:10.1158/1538-7445.AM2011-4468

Lactate dehydrogenase (LDH) was found to lose rapidly its catalytic activity during the process of entrapment in hydrated reversed micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in octane. It was demonstrated that this inactivation was caused by the surfactant which penetrated into the injected enzyme-containing aqueous bulk phase during the short time of mechanical stirring needed to convert the initial biphasic mixture into a monophasic reversed micellar solution. The unfavorable inactivation phenomenon could be efficiently eliminated by the addition of either NADH or pyruvate into the enzyme stock solution prior to its injection into AOT solution in octane. The catalytic activity of substrate-protected LDH in AOT reversed micelles increased with increasing water content of the micellar system, reaching the maximal level above wo = 30 when aqueous inner cores of reversed micelles grew large enough to allow unrestricted accommodation of the enzyme molecule. It is suggested that the employment of substrate-protected enzymes could represent a generally useful approach for producing highly efficient enzyme-containing reversed micellar reaction systems.

Proteins can be extracted from an aqueous phase into reversed micelles. This transfer is governed by electrostatic interactions, for protein and surfactant have to bear opposite charges. The presence of high salt concentrations diminishes the attractive interactions and can lead to expulsion of the solubilized proteins. This is used to recover extracted proteins. Larger proteins require a larger number of charged residues on their surface in order to be transferred into reversed micelles, so the larger the protein, the further the pH of maximal transfer is removed from the isoelectric point. The transfer profiles can be manipulated by micellar size and charge density at the interface. When the charge density at the interface is modified by variation of the type of head group of the cosurfactant, shifts of transfer profiles to higher or lower pH values are observed. Variation of the number and length of the tails of quaternary ammonium surfactants revealed that of the 16 surfactants tested, only didecyldimethyl ammonium chloride and trioctylmethylammonium chloride enabled transfer. There was no relation between the water content of the organic phase and the transfer properties of the surfactants tested. Because for application not only forward transfer is important, an alternative method for back transfer was tested. Exposing an enzyme containing reversed micellar solution to a temperature increase led to expulsion of aqueous phase and enzyme, yielding a highly concentrated enzyme preparation.

The kinetic parameters of 20 beta-hydroxysteroid dehydrogenase were determined in aqueous solutions and in reversed micellar media composed with either an anionic, a cationic or a nonionic surfactant, at low and at high ionic strength. The velocity data were analysed in two ways: first by extrapolation to infinite concentrations of both substrates to determine 'apparent' Michaelis constants and V values, and secondly by comparison to reaction rates calculated using the model presented (see first of this series of papers in this issue of the journal). Data analysis according to the first method reveals some differences in the kinetic parameters in reversed micelles as compared to those in aqueous solution, though the kinetic parameters of the enzyme seem not to be much affected by enclosure in reversed micelles. It is shown that the changes that do occur are not caused by a shift of the intramicellar pH or by electrostatic interactions between the enzyme and the surfactant head groups. Interpretation of the data using the second method assumes that the enzyme is not affected by the enclosure in reversed micelles, and that deviations with respect to the aqueous parameters are caused by exchange phenomena between distinct aqueous droplets in the organic phase and by a high effective intramicellar substrate concentration. This model is able to predict reaction rates that agree rather well with experimentally determined rates and explains why the enzyme mechanism in reversed micelles is, at all progesterone concentrations used, the same as observed at high progesterone concentrations in aqueous solution. Furthermore it clarifies the occurrence of substrate inhibition in sodium-di(ethylhexyl)sulphosuccinate-reversed micelles and the observed low activity in Triton-reversed micelles, as arising from the high partition coefficient of progesterone and the slow rate of diffusion of progesterone into the reversed micelles. From these results, and those reported for enoate reductase (see preceding paper in this issue of the journal) it can be concluded that the theory presented before (see first of this series of papers in this issue of the journal) offers a good explanation for the observed kinetic behaviour in reversed micelles, and emphasizes the importance of exchange processes between micelles.

Enoate reductase (EC 1.3.1.31) can stereospecificially reduce a variety of α,β-unsaturated carboxylates. Its use was extended to apolar media by incorporating the enzyme into a reversed micellar medium. The kinetics of the enzyme in such a medium have been investigated using 2-methylbutenoic acid as substrate and NADH as a cofactor and compared with the reaction rates in aqueous solution. In aqueous solution the enzyme obeys a ping pong mechanism [Buhler et al. (1982) Hoppe-Seyler's Z. Physiol. Chem 363, 609–625]. In 50 mM Hepes pH = 7.0 with ionic strength of 0.05 M the Michaelis constants for NADH and 2-methylbutenoic acid are 20 μM and 6.0 mM respectively. In reversed micelles the kinetics of the reaction (Michaelis constant, maximum velocity as well as inhibitory effects) were markedly different. The rate of the enzymatic reaction of enoate reductase was studied using various concentrations of 2-methylbutenoic acid and various NADH concentrations. In reversed micelles composed of the anionic detergent sodium di(ethylhexyl)sulphosuccinate, the enzymatic reaction deviates substantially from the values in aqueous solution. Using our model (see preceding paper in this issue of the journal), all kinetics could be explained as evolving from enclosure in reversed micelles without any change in the intrinsic rate parameters of the enzyme. So the enzyme itself is unaffected by incorporation in reversed micelles, but the rate of intermicellar exchange as well as the microheterogeneity of the medium, resulting in very high local concentrations of the substrate, are the most important factors altering the reaction pattern. The effect of the composition of the reversed micellar medium was also investigated using either a nonionic or a cationic surfactant. In these solutions too, exchange and microheterogeneity of the medium proved to be the most important parameters influencing the enzymatic reaction. In all reversed micellar solutions inhibition by the enoate was observed at an overall concentration of 0.5–5 mM, implying that a concentration of substrate equal to the Km value in aqueous solution may already cause inhibition in reversed micelles. At this level no inhibition by NADH was observed. The microheterogeneity of the medium also explains this inhibition of the enzyme at relatively low 2-methylbutenoic acid concentrations.

In the literature measurements of kinetic data of enzymes in reversed micelles have been interpreted in two ways. In the first, all enzyme parameters are expressed with respect to the total volume of the reversed micellar solution. In the second, the enzymatic conversion is related only to the fraction of the volume consisting of aqueous solution (pseudophase model). In this paper equations are derived describing the rate of an enzymatic reaction for three different kinds of enzymes: enzymes obeying Michaelis-Menten kinetics, enzymes following a ping-pong bi-bi mechanism and enzymes which convert substrates according to an ordered mechanism. In deriving these equations, a distinction is made between intermicellar exchange reactions of substrate(s) and product(s) and the enzymatic reaction which takes place in the waterpool of a reversed micelle. In the description, all intrinsic rate constants of the enzyme are assumed to be independent of its environment. The rate equations show that the presence and efficiency of the intermicellar exchange reaction, which supplies the enzyme with substrate and removes product, can affect the rate of an enzymatic reaction under common experimental conditions. Whereas kinetic parameters derived from double-reciprocal plots often seem to be affected by enclosure in reversed micelles, these apparent deviations from kinetics in aqueous media can be explained by the model presented here as arising from exchange phenomena. Neither the experimentally determined maximum enzyme velocity, vmax, nor the Michaelis constants are affected by the incorporation of the enzyme in reversed micelles. The deviations of kinetic parameters from the aqueous values are shown to depend strongly on the concentration of reversed micelles, the intermicellar exchange rate and the volume fraction of water, a dependence in agreement with findings reported in the literature.

A combined enzyme system, consisting of hydrogenase, lipoamide dehydrogenase and 20β-hydroxysteroid dehydrogenase has been enclosed in reversed micelles. This system catalyzes the stereo- and site-specific enzymatic reduction of apolar, poorly water-soluble ketosteroids to their corresponding 20β-hydroxyform using an in situ NADH-regenerating enzyme system and H 2 as ultimate reductant.

20 beta-Hydroxysteroid dehydrogenase was enclosed in reversed micellar media consisting of cetyltrimethyl-ammonium bromide, hexanol, organic solvent and Hepes buffer. The influence of the composition of these media on the enzymatic reduction of the apolar steroids progesterone and prednisone was investigated by varying the water content, concentration of hexanol and type of organic solvent. By changing the water content and the type of organic solvent, the hexanol to cetyltrimethylammonium bromide ratio in the interphase can be varied. This ratio was determined by phase boundary titrations. It was found that the higher this ratio, the higher the rate of steroid conversion. From variations of the hexanol content it was concluded that the rate of steroid conversion is determined by the hydrophobicity of the steroid relative to the hydrophobicity of the continuous phase and the hydrophobicity of the interphase. The hydrophobicity of the phases was expressed in log P-values. Log P is defined as the logarithm of the partition coefficient in an octanol-water two-phase system. This enabled us to derive the following relations between the hydrophobicity values for the substrate (log Ps), for the interphase (log Pi) and for the continuous phase (log Peph): [log Pi-log Ps] must be minimal to ensure a high steroid concentration in the interphase and [log Pcph-log Ps] must be large to keep the steroid concentration in the continuous phase low. With these considerations, for any given apolar compound, a medium can be composed that gives optimal enzymatic conversion.

Catalytic acitivity and stability of cholesterol oxidase dissolved in ternary systems composed on n-hexane, isopropanol, and water were studied. The dependence of catalytic acitivity on the composition of the system revealed two maxima, in contrast to the behaviour of previously studied enzymes where a single maximum has been observed. The stability profile of cholesterol oxidase showed a single sharp maximum coinciding with the microemulsion region of the phase diagram. Both catalytic activity and the first-order inactivation rate constant of cholesterol oxidase dissolved in n-hexane/isopropanol/water ternary systems were found to decrease with decreasing temperature. This decrease was more rapid for the inactivation rate constant than for catalytic activity, the activation energies being 200 and 60 kJ mol−1, respectively. Preparative conversion of cholesterol to cholestenone catalyzed by cholesteroloxidase in n-hexane/isopropanol/water ternary systems was carried out with 100% yield. Decreased temperature and the presence of catalase were required to achieve high degrees of cholesterol conversion. A simple procedure suitable for rapid separation of the reaction product and recovery of the enzyme was developed.

Proteins are spontaneously transferred from an aqueous solution into reversed micelles, provided the aqueous phase has the proper composition. Besides the composition of the aqueous phase, the composition of the organic phase and the properties of the proteins also play a role. We studied uptake profiles of 19 proteins as a function of pH of the aqueous solution. The organic phase consisted of trioctylmethylammonium chloride and nonylphenol pentaethoxylate (Rewopal HV5) as surfactant, octanol as cosurfactant and isooctane as continuous phase. In all cases, except for rubredoxin, proteins were transferred at pH values above their isoelectric point. The pH where maximal solubilization takes place can be described by the relationship: pHoptimum = isoelectric point f0.11 x 1O-j M, -0.97. So, the larger the protein, the more charge is needed to provide the energy required for the adaptation of the micellar size to the protein size. For protein transfer into sodium di-(2-ethylhexyl)sulphosuccinate (AOT) reversed micelles a similar relationship was found. The percentage of protein transferred could be related to the symmetry of charge distribution over the protein. This symmetry was expressed as the YO of random electric moments on a protein that is larger than the effective electric moment of the protein (YO S) [Barlow, D. J. and Thornton, J. M. (1986) Biopolymers 25, 17171. The larger the value of YO S, the more homogeneously the charges are distributed and the lower the percentage transfer.

Background Alzheimer’s disease (AD) is the most common neurodegenerative disease. In addition to the occurrence of amyloid deposits and widespread tau pathology, AD is associated with a neuroinflammatory response characterized by the activation of microglia and astrocytes. Protein kinase 2 (CK2, former casein kinase II) is involved in a wide variety of cellular processes. Previous studies on CK2 in AD showed controversial results, and the involvement of CK2 in neuroinflammation in AD remains elusive. Methods In this study, we used immunohistochemical and immunofluorescent staining methods to investigate the localization of CK2 in the hippocampus and temporal cortex of patients with AD and non-demented controls. We compared protein levels with Western blotting analysis, and we investigated CK2 activity in human U373 astrocytoma cells and human primary adult astrocytes stimulated with IL-1β or TNF-α. Results We report increased levels of CK2 in the hippocampus and temporal cortex of AD patients compared to non-demented controls. Immunohistochemical analysis shows CK2 immunoreactivity in astrocytes in AD and control cases. In AD, the presence of CK2 immunoreactive astrocytes is increased. CK2 immunopositive astrocytes are associated with amyloid deposits, suggesting an involvement of CK2 in the neuroinflammatory response. In U373 cells and human primary astrocytes, the selective CK2 inhibitor CX-4945 shows a dose-dependent reduction of the IL-1β or TNF-α induced MCP-1 and IL-6 secretion. Conclusions This data suggests that CK2 in astrocytes is involved in the neuroinflammatory response in AD. The reduction in pro-inflammatory cytokine secretion by human astrocytes using the selective CK2 inhibitor CX-4945 indicates that CK2 could be a potential target to modulate neuroinflammation in AD. Electronic supplementary material The online version of this article (doi:10.1186/s12974-015-0470-x) contains supplementary material, which is available to authorized users.

The effect of riboflavin and riboflavin binding proteins on the light-induced formation of reactive oxygen species and sunstruck off-flavour was studied in model beer solutions. Under model beer conditions (pH 4.0, 1 ppm riboflavin, 5% ethanol and traces of O 2 ) hydroxyl and hydroxyethyl radicals were formed upon illumination. Radical formation was measured with the spin traps N-t-butyl-α-phenylnitrone (PBN) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). DMPO appeared to be a better spin trap than PBN for studying the effect of light exposure, since PBN is photochemically active by itself. Addition of isohumulones to the model beer reduced the amount of riboflavin-induced radicals. Two different riboflavin binding proteins were tested both for their ability to scavenge riboflavin and how in turn this influenced free radical formation. The apoform of egg white riboflavin binding protein (RfBP) was more efficient in reducing radical formation than an apo-flavodoxin protein isolated from Azotobacter vinelandii. Organoleptic assessment clearly indicated that the addition of apo-RfBP to model beer solutions, containing stiochiometric amounts of riboflavin as well as isohumulones and cysteine, reduced sunstruck off-flavour formation. The dual role of riboflavin and ethanol as radical propagators in oxidiatve flavour change is discussed.