Characterization of a novel amylolytic enzyme encoded by a gene from a soil-derived metagenomic library

It has been estimated that less than 1% of the microorganisms in nature can be cultivated by conventional techniques. Thus, the classical approach of isolating enzymes from pure cultures allows the analysis of only a subset of the total naturally occurring microbiota in environmental samples enriched in microorganisms. To isolate useful microbial enzymes from uncultured soil microorganisms, a metagenome was isolated from soil samples, and a metagenomic library was constructed by using the pUC19 vector. The library was screened for amylase activity, and one clone from among approximately 30,000 recombinant Escherichia coli clones showed amylase activity. Sequencing of the clone revealed a novel amylolytic enzyme expressed from a novel gene. The putative amylase gene (amyM) was overexpressed and purified for characterization. Optimal conditions for the enzyme activity of the AmyM protein were 42 degrees C and pH 9.0; Ca2+ stabilized the activity. The amylase hydrolyzed soluble starch and cyclodextrins to produce high levels of maltose and hydrolyzed pullulan to panose. The enzyme showed a high transglycosylation activity, making alpha-(1, 4) linkages exclusively. The hydrolysis and transglycosylation properties of AmyM suggest that it has novel characteristics and can be regarded as an intermediate type of maltogenic amylase, alpha-amylase, and 4-alpha-glucanotransferase.     

Design and Synthesis of a Cell-Permeable,Drug-Like Small Molecule Inhibitor Targeting the Polo-Box Domain of Polo-Like Kinase 1

Background

Polo-like kinase-1 (Plk1) plays a crucial role in cell proliferation and the inhibition of Plk1 has been considered as a potential target for specific inhibitory drugs in anti-cancer therapy. Several research groups have identified peptide-based inhibitors that target the polo-box domain (PBD) of Plk1 and bind to the protein with high affinity in in vitro assays. However, inadequate proteolytic resistance and cell permeability of the peptides hinder the development of these peptide-based inhibitors into novel therapeutic compounds.

Methodology/Principal Findings

In order to overcome the shortcomings of peptide-based inhibitors, we designed and synthesized small molecule inhibitors. Among these molecules, bg-34 exhibited a high binding affinity for Plk1-PBD and it could cross the cell membrane in its unmodified form. Furthermore, bg-34-dependent inhibition of Plk1-PBD was sufficient for inducing apoptosis in HeLa cells. Moreover, modeling studies performed on Plk1-PBD in complex with bg-34 revealed that bg-34 can interact effectively with Plk1-PBD.

Conclusion/Significance

We demonstrated that the molecule bg-34 is a potential drug candidate that exhibits anti-Plk1-PBD activity and possesses the favorable characteristics of high cell permeability and stability. We also determined that bg-34 induced apoptotic cell death by inhibiting Plk1-PBD in HeLa cells at the same concentration as PEGylated 4j peptide, which can inhibit Plk1-PBD activity 1000 times more effectively than bg-34 can in in vitro assays. This study may help to design and develop drug-like small molecule as Plk1-PBD inhibitor for better therapeutic activity.     

Effect of matrices on affinity purification of protein C

One of the critical problems in scale-up of affinity chromatography is the mechanical strength of the support matrix against pressure. Because the costs of both the gel matrix and the ligand for the affinity chromatography are very high, the reusability of gel matrices is directly related to the total production cost. In certain cases, where the source material is viscous (e.g., blood plasma), irreversible deformation of gel matrices can readily occur, necessitating severe constraints in the flow rate. Consequently, productivity is low.We have characterized the system parameters and investigated the performance of various matrices that are commercially available. The experimental system used for this study was the immunoaffinity purification of protein C (an anticoagulant protein) from human blood plasma. The support matrices studied were cross-linked agarose, polymethyl acrylic, cellulose, and polyvinyl alcohol polymers. The major system parameters studied were pressure tolerance, coupling efficiency, adsorption efficiency, and batch adsorption/desorption kinetics of protein C to/from the monoclonal antibody (MAb)-Matrix complex. In addition, the apparent equilibrium constant and bandwidth of the product concentration profile in the eluate were characterized by performing pulse tests.A methodology was developed for evaluating the immunoaffinity colum performance for the separation of protein C. By utilizing the experimentally measured parameters, the flow rate limitation for each purification step was computed. Then, the purification performance of the matrices were evaluated in terms of productivity per unit time. Among the matrices tested, cellulose was superior in overall performance for the immunoaffinity purification of protein C using a 10 cm x 10 cm column.     

Complete mitochondrial genome of the Baikal teal Anas formosa (Aves, Anseriformes, Anatidae)

The Baikal teal Anas formosa (Aves, Anseriformes, Anatidae) is classified as "Vulnerable" on the IUCN Red List. Here, whole mitochondrial genome of A. formosa was amplified and sequenced. The total length of the Baikal teal mitochondrial genome is 16,594?bp, which consists of 13 protein-coding, 2 rRNA, 22 tRNA genes and 1 control region. The characteristics of the mitochondrial genomes were analyzed and discussed in detail.     

Trp-Lys-Tyr-Met-Val-Met activates mitogen-activated protein kinase via a PI-3 kinase-mediated pathway independent of PKC

Trp-Lys-Tyr-Met-Val-Met (WKYMVM) is a novel potent peptide which can stimulate phosphoinositide hydrolysis in U937 as well as U266 and HL-60 cells (Baek et al., J. Biol. Chem. 271, 8170 (1996)). The peptide also induces superoxide generation in human neutrophils (Seo et al., J. Immunol. 158, 1896 (1997)). However, the signaling pathway down-stream of PLC set in motion by the peptide is not yet completely understood. We studied the signaling pathway of the peptide with the goal of elucidating the mechanism of the peptide's action. WKYMVM induced a rapid and transient activation of the ERKs in human histiocytic lymphoma cells, U937. The ERK1 activation peaked at 5 min and returned to the basal level after 30 min. The ERK1 stimulation by the peptide was partially inhibited by pretreatment of the cells with pertussis toxin (PTX), implicating G-protein involvement in the peptide's action. Pretreatment of staurosporine, protein kinase C (PKC) inhibitor, or PKC down-regulating PMA had no impact on the ERK1 activation by the peptide, indicating that the signaling pathway is independent of PKC activation. Pretreatment of the cells with neomycin and intracellular Ca2+ mobilizing reagents had also no effect on the ERK1 activation by the peptide. However, pretreatment with wortmannin or LY294002, the inhibitor of phosphatidylinositol 3 kinase (PI-3K), strongly inhibited peptide-stimulated ERK1 activation. Our results suggest that PI-3K may be an important participant in the ERK cascade induced by the peptide. Furthermore, the treatment of U937 cells with the peptide activated p74Raf-1, an upstream kinase of ERK. Taken together, our results suggest that the peptide activate ERK via a G-protein/PI-3K/Ras/Raf-1 mediated signaling pathway in U937 cells.     

Delphinidin prevents hypoxia-induced mouse embryonic stem cell apoptosis through reduction of intracellular reactive oxygen species-mediated activation of JNK and NF-κB, and Akt inhibition

Delphinidin, gallic acid, betulinic acid, and ursolic acid, which are bio-active ingredients in a variety of fruits, vegetables, and herbs, have potent antioxidant activity and various biological activities. However, it is not clear whether these bio-active ingredients can significantly contribute to the protection of embryonic stem (ES) cells from hypoxia-induced apoptosis. In the present study, hypoxia-induced ES cells apoptosis with time, which were abrogated by pretreatment with all ingredients. Hypoxia-induced ROS generation was blocked by pretreatment with all ingredients in a dose-dependent manner, with the maximum ROS scavenging effect observed for delphinidin. Hypoxia increased phosphorylation of JNK and NF-κB were blocked by pretreatment of delphinidin as well as NAC. Hypoxia decreased phosphorylation of Akt^thr308 and ^ser473; these decreases were reversed by pretreatment with delphinidin or NAC. However, Akt inhibition did not affect NF-κB phosphorylation. Delphinidin attenuated the hypoxia-induced increase in Bax, cleaved caspase-9, cleaved caspase-3, and decrease in Bcl-2, which were diminished by pretreatment of Akt inhibitor. Hypoxia induced Bax translocation from the cytosol to mitochondria. Furthermore, hypoxia induced mitochondria membrane potential loss and cytochrome c release in cytosol, which were blocked by delphinidin pretreatment. Hypoxia induced cleavage of procaspase-9 and procaspase-3 which were blocked by delphinidin or SP600125, but Akt inhibitor abolished the protection effect of delphinidin. Moreover, inhibition of JNK and NF-κB abolished hypoxia-induced ES cell apoptosis and inhibition of Akt attenuated delphinidin-induced blockage of apoptosis. The results indicate that delphinidin can prevent hypoxia-induced apoptosis of ES cells through the inhibition of JNK and NF-κB phosphorylation, and restoration of Akt phosphorylation.