Arabidopsis HY5 protein functions as a DNA-binding tag for purification and functional immobilization of proteins on agarose/DNA microplate

Protein microarray is considered to be one of the key analytical tools for high-throughput protein function analysis. Here, we report that the Arabidopsis HY5 functions as a novel DNA-binding tag (DBtag) for proteins. We also demonstrate that the DBtagged proteins could be immobilized and purified on a newly designed agarose/DNA microplate. Furthermore, we show three applications using the microarray: (1) detection of autophosphorylation activity of DBtagged human protein kinases and inhibition of their activity by staurosporine, (2) specific cleavage of DBtagged proteins by a virus protease and caspase 3, and (3) detection of a protein-protein interaction between the DBtagged UBE2N and UBE2v1. Thus, this method may facilitate rapid functional analysis of a wide range of proteins.     

A fusion protein between rac and p67phox (1-210) reconstitutes NADPH oxidase with higher activity and stability than the individual components.

Activation of the phagocyte NADPH oxidase, a superoxide-generating enzyme, involves assembly of cytosolic p47(phox), p67(phox), and rac with the membrane-associated cytochrome b(558). Following cell-free activation, enzymatic activity is highly labile [Tamura, M., Takeshita, M., Curnutte, J. T., Uhlinger, D. J., and Lambeth, J. D. (1992) J. Biol. Chem. 267, 7529-7538]. In an attempt to stabilize the activity and to investigate the nature of the complex, we have produced fusion proteins between rac and a C-terminal truncated form of p67(phox) (residues 1-210, 67N), which is a minimal active fragment. In a cell-free system, a fusion protein 67N-rac had higher activity and a 3-fold higher affinity than the individual cytosolic proteins, and 67N-Ser3-rac, which has a longer linker, showed a similar activity with the individual proteins. In contrast, rac-67N, a fusion in the opposite orientation, showed considerably lower activity. The enzyme activity reconstituted with 67N-rac showed a 10-fold higher stability and a lower K(m) for NADPH than the individual components. In the absence of p47, 67N-rac fusion protein at a high concentration showed nearly full activation, which was higher than that with the individual components. These results indicate that covalent binding between p67N and rac in the correct order produces a more stable complex than the individual components, suggesting that interactions among the subunits significantly influence the duration of the oxidase activation. On the basis of these findings, we propose a model for the topology among rac, 67N, and cytochrome b(558).     

Possible participation of intracellular platelet-activating factor in NF-κB activation in rat peritoneal macrophages

As we had found previously that thapsigargin, an endomembrane Ca²⁺-ATPase inhibitor, induces production of intracellular platelet-activating factor (PAF) [Br. J. Pharmacol. 116 (1995) 2141], we decided to investigate the possible roles of intracellular PAF in nuclear factor (NF)-κB activation of thapsigargin-stimulated rat peritoneal macrophages. When rat peritoneal macrophages were stimulated with thapsigargin, the level of inhibitory protein of NF-κB-α (IκB-α) was decreased and the nuclear translocation of NF-κB was increased. The thapsigargin-induced activation of NF-κB was inhibited by the PAF synthesis inhibitor SK&F 98625 and the PAF antagonist E6123. Structurally unrelated PAF antagonists such as E5880 and L-652,731 also inhibited the thapsigargin-induced activation of NF-κB. Lipopolysaccharide (LPS)-induced activation of NF-κB was also suppressed by these drugs. In a culture of rat peritoneal macrophages, exogenously added PAF did not induce degradation of IκB-α. These findings suggest that the intracellular PAF produced by the stimulation with thapsigargin or LPS is involved in activation of the NF-κB pathway.     

Accumulation of Phenylpropanoids in the Cotyledons of Morning Glory (Pharbitis nil) Seedlings during the Induction of Flowering by Low Temperature Treatment, and the Effect of Precedent Exposure to High-Intensity Light

Extracts from the cotyledons of seedlings of Pharbitis nil strain‘Violet’ cultured at low temperature, which inducestheir flowering even in continuous light, with or without precedentexposure to high-intensity light, which shortens the periodof low temperature required for flowering, were analyzed byHPLC for substances correlating with the flower-inducing process.The content of two phenylpropanoids were found to increase duringthe low-temperature, and were identified as 3-O-feruloylquinicacid and dehydrodiconiferyl alcohol-13-O-ß-D-glucoside.The increase was more rapid in the cotyledons exposed to high-intensitylight before the low-temperature. This suggests that the accumulationof these compounds is correlated to the promotive effect ofhigh-intensity light on the flower-induction by low temperature. (Received March 7, 1994; Accepted April 2, 1994)     

Use of live varicella vaccine in children with acute leukemia and malignant lymphoma

Live varicella vaccine was given to 7 children with acute leukemia and 1 child with non-Hodgkin's lymphoma. Seroconversion without any clinical reactions was observed in 4 children in remission from acute leukemia without interruption of anticancer medication. These children have been free from varicella for 5 to 11 months after vaccination. Mild to severe clinical reactions developed in 3 of 4 children who were receiving remission induction chemotherapy. It is concluded that live varicella vaccine is effective in children with acute leukemia without interrupting anticancer medication, but that it is not suitable for children with leukemia and lymphoma who are receiving remission induction chemotherapy with antileukemic agents.     

Recursive Random Lasso (RRLasso) for Identifying Anti-Cancer Drug Targets

Uncovering driver genes is crucial for understanding heterogeneity in cancer. L ₁-type regularization approaches have been widely used for uncovering cancer driver genes based on genome-scale data. Although the existing methods have been widely applied in the field of bioinformatics, they possess several drawbacks: subset size limitations, erroneous estimation results, multicollinearity, and heavy time consumption. We introduce a novel statistical strategy, called a Recursive Random Lasso (RRLasso), for high dimensional genomic data analysis and investigation of driver genes. For time-effective analysis, we consider a recursive bootstrap procedure in line with the random lasso. Furthermore, we introduce a parametric statistical test for driver gene selection based on bootstrap regression modeling results. The proposed RRLasso is not only rapid but performs well for high dimensional genomic data analysis. Monte Carlo simulations and analysis of the “Sanger Genomics of Drug Sensitivity in Cancer dataset from the Cancer Genome Project” show that the proposed RRLasso is an effective tool for high dimensional genomic data analysis. The proposed methods provide reliable and biologically relevant results for cancer driver gene selection.     

Robust Prediction of Anti-Cancer Drug Sensitivity and Sensitivity-Specific Biomarker

The personal genomics era has attracted a large amount of attention for anti-cancer therapy by patient-specific analysis. Patient-specific analysis enables discovery of individual genomic characteristics for each patient, and thus we can effectively predict individual genetic risk of disease and perform personalized anti-cancer therapy. Although the existing methods for patient-specific analysis have successfully uncovered crucial biomarkers, their performance takes a sudden turn for the worst in the presence of outliers, since the methods are based on non-robust manners. In practice, clinical and genomic alterations datasets usually contain outliers from various sources (e.g., experiment error, coding error, etc.) and the outliers may significantly affect the result of patient-specific analysis. We propose a robust methodology for patient-specific analysis in line with the NetwrokProfiler. In the proposed method, outliers in high dimensional gene expression levels and drug response datasets are simultaneously controlled by robust Mahalanobis distance in robust principal component space. Thus, we can effectively perform for predicting anti-cancer drug sensitivity and identifying sensitivity-specific biomarkers for individual patients. We observe through Monte Carlo simulations that the proposed robust method produces outstanding performances for predicting response variable in the presence of outliers. We also apply the proposed methodology to the Sanger dataset in order to uncover cancer biomarkers and predict anti-cancer drug sensitivity, and show the effectiveness of our method.     

Densification of cyanobacteria from a lake leading to production of β‐cyclocitral and related volatile organic compounds and species change

The purpose of the present study was to demonstrate that the lysis with the blue color formation was caused by densification of the cyanobacteria, and related events of the species change in the cyanobacteria were induced by the resulting volatile organic compounds (VOCs), particularly β‐cyclocitral. In order to obtain a high cell density of cyanobacteria in the laboratory, a concentration technique (graduated cylinder method) using the buoyancy of the gas vesicles was successfully used. The collected scum contained mainly Dolichospermum spp. and Microcystis, and the dispersed cyanobacteria were concentrated in the surface layer after several hours and the concentration ratio became approximately 10. The concentrated cyanobacteria were gradually lysed, while some of the cyanobacteria sank to the bottom, which finally died and disappeared. This method has the additional advantage that it is possible to visualize the entire lysis process. During the concentration process, β‐cyclocitral and its oxidation products together with β‐ionone were significantly detected. Because β‐cyclocitral was easily oxidized to the corresponding carboxylic acid, the pH of the water in the graduated cylinder decreased to approximately 6. Under favorable conditions, lysis with the blue color from phycocyanin could be observed due to the acid stress. Overall, the results of the present study were consistent with the hypothesis that VOCs were produced when the cyanobacteria are highly dense, and that the lysis with the blue color formation occurs due to the higher density.