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Apoptosis signal-regulating kinase 1 (ASK1) is a mediator of the MAPK signaling cascade, which regulates different cellular processes including apoptosis, cell survival, and differentiation. The increased activity of ASK1 is associated with a number of human diseases and this protein kinase is considered as promising therapeutic target. In the present study, the kinase domain of human ASK1 was expressed in Escherichia coli ( E. coli ) in soluble form. The expression level of ASK1 was around 0.3–0.47 g per 1 L after using auto-induction protocol or IPTG induction. A one-step on column method for the efficient purification of recombinant ASK1 was performed. Our approach yields sufficient amount of recombinant ASK1, which can be used for inhibitor screening assays and different crystallographic studies. [ABSTRACT FROM AUTHOR]

The effect of various N,N ′-substituents in the molecule of benzothiazole trimethine cyanine dye on its ability to sense the amyloid aggregates of protein was studied. The dyes are low fluorescent when free and in the presence of monomeric proteins, but their emission intensity sharply increases in complexes with aggregated insulin and lysozyme, with the fluorescence quantum yield reaching up to 0.42. The dyes carrying butyl, hydroxyalkyl, and phenylalkyl groups as N,N ′-substituents possess the increased fluorescent sensitivity to fibrillar lysozyme, whereas the ones carrying quaternary amino groups are preferably sensitive to fibrillar insulin. This fluorescent sensitivity preference provided by the N,N ′-functional groups could be explained by the interaction between these groups and protein side chains. The strongest fluorescent response (up to 70 times) and the same sensitivity to aggregates of both proteins were exhibited by the dye D-51 carrying N -sulfoalkyl group. The studied cyanines allow the detection of fibrillar aggregates in the wide range up to 0.8 to 300 μg/ml and permit monitoring the protein aggregation kinetics with high reproducibility. The modification of trimethine cyanine dyes by functional substituents in N,N ′-positions is suggested as a tool for the design of fluorescent molecules with the enhanced fluorescent sensitivity to the fibrillar aggregates of proteins. [ABSTRACT FROM AUTHOR]

Abstract: An electrochemical method based on the bioorganometallic Fc-ATP cosubstrate for kinase-catalyzed phosphorylation reactions was used for monitoring casein kinase 2 (CK2) phosphorylations in the absence and presence of five indole/quinolone-based potential inhibitors. Fc-phosphorylation of immobilized peptide RRRDDDSDDD on Au surfaces resulted in a current density at approximately 460±10mV. An electrochemical redox signal was significantly decreased in the presence of inhibitors. In addition, the electrochemical signal was concentration dependent with respect to the potential inhibitors 1 to 5, which proved to be viable CK2 drug targets with estimated IC50 values in the nanomolar range. [Copyright &y& Elsevier]

Abstract: Kinetics and equilibria of cyanine dyes thiazole orange (TO) and benzothiazole orange (BO) self-aggregation and binding to CT-DNA are investigated in aqueous solution at 25°C and pH 7. Absorbance spectra and T-jump experiments reveal that BO forms J-aggregates while TO forms more stable H-aggregates. Fluorescence and absorbance titrations show that TO binds to DNA more tightly than BO. TO stacks externally to DNA for low polymer-to-dye concentration ratios (C P/C D) while dye intercalation occurs for high values of C P/C D. T-jump and stopped-flow experiments performed at high C P/C D agree with reaction scheme D+S⇆D,S⇆DSI ⇆DSII where the precursor complex D,S evolves to a partially intercalated complex DSI which converts to the more stable intercalate DSII. Non-electrostatic forces play a major role in D,S stabilization. Last step is similar for both dyes suggesting accommodation of the common benzothiazole residue between base pairs. Experiments using poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC) confirm base pair preference for TO. [Copyright &y& Elsevier]

Abstract: A T-jump investigation of the binding of Cyan40 [3-methyl-2-(1,2,6-trimethyl-4(1H)pyridinylidenmethyl)-benzothiazolium ion] and CCyan2 [3-methyl-2-[2-methyl-3-(3-methyl-2(3H)-benzothiazolylidene)-1-propenyl]-benzothiazolium ion] with poly(dA–dT)·poly(dA–dT) and poly(dG–dC)·poly(dG–dC) is performed at I =0.1M (NaCl), 25°C and pH 7. Two kinetic effects are observed for both systems. The binding process is discussed in terms of the sequence D+P⇆P,D⇆PDI ⇆PDII, which leads first to fast formation of a precursor complex P,D and then to a partially intercalated complex PDI which converts to the fully intercalate complex PDII. Concerning CCyan2 the rate parameters depend on the polymer nature and their analysis shows that in the case of poly(dG–dC)·poly(dG–dC) the most stable bound form is the fully intercalated complex PDII, whereas in the case of poly(dA–dT)·poly(dA–dT) the partially intercalated complex PDI is the most stable species. Concerning Cyan40, the rate parameters remain unchanged on going from A–T to G–C indicating that this dye is unselective. [Copyright &y& Elsevier]