Phenomena Affecting the Equilibrium of Al(III) and Zn(II) Extraction with Winsor II Microemulsions

The extraction of Al(III) and Zn(II) from an aqueous solution with two water-in-oil microemulsions, one containing di(2-ethylhexyl)phosphoric acid (DEHPA), was investigated to aid the understanding of the role of the extractant and the metal specific characteristics in the mechanism of microemulsion extraction. The extraction of Al with the DEHPA microemulsion increased by a factor of about 10 with respect to that in the conventional DEHPA system, whereas the extraction of Zn was lower than that in the single DEHPA system. Extraction with the DEHPA-free microemulsion was very low, showing that metal ion solubilization was not important in the mechanism of microemulsion extraction. It is proposed that the effect of the mixed microemulsion on the metal distribution coefficient is the result of the balance between a decrease in the complexation reaction yield due to the interaction between butanol and DEHPA, and the adsorption of the metal complex at the macro- and microinterfaces. The former leads to a decrease in Zn(II) extraction and the latter to Al(III) extraction synergism. Copyright 2000 Academic Press.     

Characterization of hydromedusan Ca2+-regulated photoproteins as a tool for measurement of Ca2+concentration

Calcium ion is a ubiquitous intracellular messenger, performing this function in many eukaryotic cells. To understand calcium regulation mechanisms and how disturbances of these mechanisms are associated with disease states, it is necessary to measure calcium inside cells. Ca²⁺-regulated photoproteins have been successfully used for this purpose for many years. Here we report the results of comparative studies on the properties of recombinant aequorin from Aequorea victoria, recombinant obelins from Obelia geniculata and Obelia longissima, recombinant mitrocomin from Mitrocoma cellularia, and recombinant clytin from Clytia gregaria as intracellular calcium indicators in a set of identical in vitro and in vivo experiments. Although photoproteins reveal a high degree of identity of amino acid sequences and spatial structures, and, apparently, have a common mechanism for the bioluminescence reaction, they were found to differ in the Ca²⁺ concentration detection limit, the sensitivity of bioluminescence to Mg²⁺, and the rates of the rise of the luminescence signal with a sudden change of Ca²⁺ concentration. In addition, the bioluminescence activities of Chinese hamster ovary cells expressing wild-type photoproteins also differed. The light signals of cells expressing mitrocomin, for example, slightly exceeded the background, suggesting that mitrocomin may be hardly used to detect intracellular Ca²⁺ without modifications improving its properties. On the basis of experiments on the activation of endogenous P2Y₂ receptor in Chinese hamster ovary cells by ATP, we suggest that wild-type aequorin and obelin from O. longissima are more suitable for calcium detection in cytoplasm, whereas clytin and obelin from O. geniculata can be used for calcium measurement in cell compartments with high Ca²⁺ concentration. Figure

Hydromedusan photoproteins differ in Ca²⁺ concentration detection limit, sensitivity of bioluminescence to Mg²⁺, and rates of rise of luminescence signal with a sudden change of [Ca²⁺] despite a high degree of identity of their amino acid sequences and spatial structures, and, apparently, a common mechanism for the bioluminescence reaction.     

New approach to the defibrillation problem: Suppression of the spiral wave activity of cardiac tissue

A model of an excitable medium is considered for describing the development of fibrillation (i.e., spatiotemporal chaos) in cardiac tissue through the generation of a set of coexisting spiral waves. It is shown that a weak external point action on such a medium leads to the suppression of all spiral waves and, correspondingly, to the stabilization of the system dynamics. After reaching the regular regime, only the external source exists in the medium. The frequencies and amplitudes at which such stabilization occurs are determined. The case of the action of several point sources is considered. Analysis is performed using the Bray method to identify the number of spiral waves.     

Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications

Copepod luciferases—a family of small secretory proteins of 18.4–24.3 kDa, including a signal peptide—are responsible for bright secreted bioluminescence of some marine copepods. The copepod luciferases use coelenterazine as a substrate to produce blue light in a simple oxidation reaction without any additional cofactors. They do not share sequence or structural similarity with other identified bioluminescent proteins including coelenterazine‐dependent Renilla and Oplophorus luciferases. The small size, strong luminescence activity and high stability, including thermostability, make secreted copepod luciferases very attractive candidates as reporter proteins which are particularly useful for nondisruptive reporter assays and for high‐throughput format. The most known and extensively investigated representatives of this family are the first cloned GpLuc and MLuc luciferases from copepods Gaussia princeps and Metridia longa, respectively. Immediately after cloning, these homologous luciferases were successfully applied as bioluminescent reporters in vivo and in vitro, and since then, the scope of their applications continues to grow. This review is an attempt to systemize and critically evaluate the data scattered through numerous articles regarding the main structural features of copepod luciferases, their luminescent and physicochemical properties. We also review the main trends of their application as bioluminescent reporters in cell and molecular biology.     

机械泵驱动两相回路的实时动态模型分析

在航空航天领域,为了加速系统设计及测试进度,通常需要进行半实物实时仿真,即控制器用实物,受控对象采用数学模型。本文开发出了基于Matlab/Simulink的两相传热模块,并用其搭建了某机械泵驱动两相回路的实时动态模型。通过与实验的对比,验证了模型的可靠性,表明该模型满足实时要求,可以在下一步用于半实物仿真。     

Tyr72 and Tyr80 are Involved in the Formation of an Active Site of a Luciferase of Copepod Metridia longa

Luciferase of copepod Metridia longa (MLuc) is a naturally secreted enzyme catalyzing the oxidative decarboxylation of coelenterazine with the emission of light. To date, three nonallelic isoforms of different lengths (17–24 kDa) for M. longa luciferase have been cloned. All the isoforms are single‐chain proteins consisting of a 17‐residue signal peptide for secretion, variable N‐terminal part and conservative C‐terminus responsible for luciferase activity. In contrast to other bioluminescent proteins containing a lot of aromatic residues which are frequently involved in light emission reaction, the C‐terminal part of MLuc contains only four Phe, two Tyr, one Trp and two His residues. To figure out whether Tyr residues influence bioluminescence, we constructed the mutants with substitution of Tyr to Phe (Y72F and Y80F). Tyrosine substitutions do not eliminate the ability of luciferase to bioluminescence albeit significantly reduce relative specific activity and change bioluminescence kinetics. In addition, the Tyr replacements have no effect on bioluminescence spectrum, thereby indicating that tyrosines are not involved in the emitter formation. However, as it was found that the intrinsic fluorescence caused by Tyr residues is quenched by a reaction substrate, coelenterazine, in concentration‐dependent manner, we infer that both tyrosine residues are located in the luciferase substrate‐binding cavity.     

Coelenterazine-v ligated to Ca2+-triggered coelenterazine-binding protein is a stable and efficient substrate of the red-shifted mutant of Renilla muelleri luciferase

It has been shown that the coelenterazine analog, coelenterazine-v, is an efficient substrate for a reaction catalyzed by Renilla luciferase. The resulting bioluminescence emission maximum is shifted to a longer wavelength up to 40 nm, which allows the use of some “yellow” Renilla luciferase mutants for in vivo imaging. However, the utility of coelenterazine-v in small-animal imaging has been hampered by its instability in solution and in biological tissues. To overcome this drawback, we ligated coelenterazine-v to Ca²⁺-triggered coelenterazine-binding protein from Renilla muelleri, which apparently functions in the organism for stabilizing and protecting coelenterazine from oxidation. The coelenterazine-v bound within coelenterazine-binding protein has revealed a greater long-term stability at both 4 and 37 °C. In addition, the coelenterazine-binding protein ligated by coelenterazine-v yields twice the total light over free coelenterazine-v as a substrate for the red-shifted R. muelleri luciferase. These findings suggest the possibility for effective application of coelenterazine-v in various in vitro assays.     

Luminescence Activity Decreases When v-coelenterazine Replaces Coelenterazine in Calcium-Regulated Photoprotein—A Theoretical and Experimental Study

Calcium-regulated photoproteins are found in at least five phyla of organisms. The light emitted by those photoproteins can be tuned by mutating the photoprotein and/or by modifying the substrate coelenterazine (CTZ). Thirty years ago, Shimomura observed that the luminescence activity of aequorin was dramatically reduced when the substrate CTZ was replaced by its analog v-CTZ. The latter is formed by adding a phenyl ring to the π-conjugated moiety of CTZ. The decrease in luminescence activity has not been understood until now. In this paper, through combined quantum mechanics and molecular mechanics calculations as well as molecular dynamics simulations, we discovered the reason for this observation. Modification of the substrate changes the conformation of nearby aromatic residues and enhances the π-π stacking interactions between the conjugated moiety of v-CTZ and the residues, which weakens the charge transfer to form light emitter and leads to a lower luminescence activity. The microenvironments of CTZ in obelin and in aequorin are very similar, so we predicted that the luminescence activity of obelin will also dramatically decrease when CTZ is replaced by v-CTZ. This prediction has received strong evidence from currently theoretical calculations and has been verified by experiments.     

Unanimous Model for Describing the Fast Bioluminescence Kinetics of Ca2+‐regulated Photoproteins of Different Organisms

Upon binding their metal ion cofactors, Ca²⁺‐regulated photoproteins display a rapid increase of light signal, which reaches its peak within milliseconds. In the present study, we investigate bioluminescence kinetics of the entire photoprotein family. All five recombinant hydromedusan Ca²⁺‐regulated photoproteins—aequorin from Aequorea victoria, clytin from Clytia gregaria, mitrocomin from Mitrocoma cellularia and obelins from Obelia longissima and Obelia geniculata—demonstrate the same bioluminescent kinetics pattern. Based on these findings, for the first time we propose a unanimous kinetic model describing the bioluminescence mechanism of Ca²⁺‐regulated photoproteins.